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Extracção de Compostos Fenólicos com Sistemas
Aquosos Bifásicos
Extraction of Phenolic Compounds with Aqueous
Two- Phase Systems
Ana Filipa
Martins Cláudio
Da Silva
Universidade de Aveiro Departamento de Química
2010
Universidade de Aveiro Departamento de Química
2010
Ana Filipa
Martins Cláudio
Da Silva
Extracção de Compostos Fenólicos com Sistemas
Aquosos Bifásicos
Extraction of Phenolic Compounds with Aqueous
Two Phase Systems
Dissertação apresentada à Universidade de Aveiro para cumprimento
dos requisitos necessários à obtenção do grau de Mestre em
Engenharia Química, realizada sob a orientação científica do
Professor Dr. João Manuel da Costa e Araújo Pereira Coutinho,
Professor Associado com agregação do Departamento de Química da
Universidade de Aveiro, e co-orientação de Dra. Mara Guadalupe
Freire Martins, Estagiária de Pós-Doutoramento no Instituto de
Tecnologia Química e Biológica, ITQB2, Universidade Nova de
Lisboa.
Dedico este trabalho às pessoas mais importantes da minha vida: à minha mãe, ao meu pai, ao
Paulo e à mana.
o júri
presidente Prof. Doutor Dmitry Victorovitch Evtyugin Professor Associado com Agregação da Universidade de Aveiro
Prof. Doutor João Manuel da Costa e Araújo Pereira Coutinho
Professor Associado com Agregação da Universidade de Aveiro
Doutor José Manuel da Silva Simões Esperança
Investigador Auxiliar no Instituto de Tecnologia Química e Biológica,ITQB2,
Universidade Nova de Lisboa
Doutora Mara Guadalupe Freire Martins
Estagiária de Pós-Doutoramento no Instituto de Tecnologia Química e Biológica,
ITQB2, Universidade Nova de Lisboa
Agradecimentos Antes de mais, um especial agradecimento ao Professor João Coutinho, por
acreditar em mim e me ter convidado para o mundo da investigação que
desde o início tanto me fascinou, e contribuiu para o meu crescimento
profissional e pessoal. Agradeço a todos os membros dos Path (Pedro
Carvalho, Maria Jorge, Sónia Ventura, Mariana Belo, Bernd, Luciana,
Mara Freire e Jorge Pereira) e mini Path (Rita Teles, Anabel, Rute, Marta,
Vanda, Catarina Neves, Catarina Varanda, Samuel, Joel, Sílvia, Francisca,
Rita Brites e Ana Maria) pelos lanches, cafezinhos, apoio, ensinamentos e
pelo espírito de inter-ajuda. Não posso deixar de dizer um obrigado
especial à Luciana, por ter sido a primeira pessoa do grupo a trabalhar
comigo; à Mara, pelo tanto que me ensinou, corrigiu, apoiou, incentivou, e
me fez pensar, bem como pela maneira fantástica como vê as coisas,
mesmo quando todos os resultados parecem estar mal! Muito obrigado
também à Sónia e ao Jorge pelo apoio incondicional, pelas dicas, pelos
almoços, jantares, gargalhadas e ajuda a todos os níveis. Obrigado Rita
Teles e Anabel pela amizade e apoio. Um especial obrigado também à Ana
Maria pelo que me ajudou. Não posso deixar de agradecer aos “amigos de
Aveiro” (Filipe, Susana, Joana, Ana Catarina,…), aos “amigos de Ovar” e
aos “amigos mais crescidos” que foram muito importantes para mim, neste
ano, devido à compreensão, amizade e demonstração de carinho que
tiveram para comigo. Por último, e por serem as pessoas mais especiais da
minha vida, tenho que agradecer muito à minha mãe por ter aturado o meu
mau-humor, pelo apoio incondicional e por acreditar em mim; À Inês por
ser a mana mais fantástica e dedicada, e pela loiça que lavou na minha vez;
Ao meu pai e à Maria José pelo apoio e compreensão; aos meus “futuros
sogros” pelo carinho e ajuda incansável; à tia Bélinha pelas perguntas que
me faziam pensar, pelo apoio e pelos risos que me ajudavam a descontrair.
Ao meu querido namorado pelo apoio total e absoluto, por compreender
todas as minhas decisões, por nunca me ter deixado desistir, pelos choros
que aguentou, pelas gargalhadas que fez dar, pelas horas ao computador a
ajudar-me a trabalhar, por tudo! Obrigado do fundo de coração a todas
estas pessoas, ao resto da família e a outras que se cruzaram comigo neste
ano cheio de emoções que tanto me ajudou a crescer a nível profissional e
pessoal.
Palavras-chave Sistemas aquosos bifásicos, líquidos iónicos, compostos fenólicos, vanilina,
ácido gálico, extracção, coeficientes de partição, diagramas de fase.
Resumo Nos últimos anos, os sistemas aquosos bifásicos (ATPS), utilizando líquidos
iónicos, têm revelado um enorme potencial no desenvolvimento de novas
técnicas de separação e purificação de biomoléculas, mantendo as suas
características funcionais intactas.
Neste trabalho, os coeficientes de partição da vanilina e ácido gálico, dois
compostos fenólicos com aplicações e propriedades antioxidantes bem
conhecidas, foram determinados recorrendo a ATPS envolvendo líquidos
iónicos. Foram avaliadas três condições no processo de partição da vanilina:
natureza catiónica e aniónica do líquido iónico (LI), a temperatura de
equilíbrio e a concentração de vanilina adicionada ao sistema. Todos os
parâmetros demonstraram influenciar a partição da vanilina entre as duas fases
aquosas. Para obter informação termodinâmica sobre o processo de partição,
foram determinadas as funções termodinâmicas molares de transferência da
vanilina. Os resultados indicaram que a partição da vanilina resulta
essencialmente de um balanço de contribuições entálpicas e entrópicas, onde
aniões e catiões mais complexos do LI desempenham um papel crucial. Foram
também determinadas duas propriedades termofísicas para estes sistemas,
viscosidade e densidade, às mesmas composições às quais se determinaram os
coeficientes de partição.
Na partição do ácido gálico, foram avaliados diferentes LIs e a influência do
pH do meio aquoso por adição de sais inorgânicos distintos. Estes dois
parâmetros demonstraram influenciar fortemente a capacidade de extracção
dos ATPS estudados. Dada a escassez dos diagramas de fase envolvendo
sistemas aquosos de LIs e Na2SO4, os diagramas ternários correspondentes a
cada LI, e respectivas tie-lines e comprimentos destas, foram também
determinados a 298 K. Em todos os sistemas estudados e em todas as
condições testadas, tanto a vanilina como o ácido gálico, mostraram sofrer
uma migração preferencial para a fase rica em LI. Com base neste trabalho,
pode-se afirmar que os novos ATPS propostos apresentam uma elevada
eficiência de extracção para compostos fenólicos constituindo assim uma nova
plataforma para processos de separação.
Keywords Aqueous two-phase systems, ionic liquids, phenolic compounds, vanillin,
gallic acid, extraction, partition coefficients, phase diagrams.
Abstract In recent years, ionic-liquid-based aqueous two-phase systems (ATPS)
have been object of great interest due to their potential for the design of
new “green” separation processes, in particular for the purification and
separation of biomolecules, maintaining their functional characteristics
unchanged.
In this work, the partition coefficients of vanillin and gallic acid, two well
known phenolic compounds, were determined using improved ionic-liquid-
based ATPS. Three parameters were evaluated in the vanillin partitioning
process: the ionic liquid (IL) cation and anion nature, the temperature of
equilibrium and the concentration of vanillin in the system. All parameters
have shown to influence the vanillin partitioning. In an attempt to elucidate
the thermodynamics of the partitioning process, the standard molar
thermodynamic functions of transfer of vanillin were also determined. The
results indicated that the partition of vanillin results from an interplay
between enthalpic and entropic contributions where both the IL anion and
more complex cations play an essential role. Moreover, viscosities and
densities of both aqueous phases were experimentally measured at the
mass fraction compositions for which the partition coefficients were
determined.
Regarding the partitioning of gallic acid, different ILs and the influence of
the aqueous medium pH, achieved by the addition of distinct inorganic
salts, were evaluated. These two parameters have shown to strongly
influence the extraction ability by IL-based ATPS. Due to the lack of the
ternary phase diagrams compositions containing ILs and the salt Na2SO4,
the respective individual phase diagrams, tie-lines and tie-line lengths,
were additionally determined at 298 K.
In all systems and conditions tested, both vanillin and gallic acid
preferentially migrated to the IL-rich phase. The new proposed ATPS
present large extraction efficiencies for phenolic compounds and represent
a new platform for separation techniques.
I
Contents
Contents ......................................................................................................................................... I
List of Tables .............................................................................................................................. III
List of Figures ............................................................................................................................. IV
Notation ...................................................................................................................................... VI
List of symbols ....................................................................................................................... VI
List of Abbreviations ............................................................................................................. VII
1. Introduction .............................................................................................................................. 1
1.1. Scopes and Objectives ...................................................................................................... 2
1.2. Ionic Liquids ..................................................................................................................... 3
1.3. Extraction of Biomolecules Using Aqueous Two-Phase Systems (ATPS)........................ 6
1.4. Phenolic compounds (PhCs) ............................................................................................. 8
2. Extraction of vanillin in ATPS with ILs ................................................................................. 12
2.1. Vanillin ........................................................................................................................... 13
2.2. Experimental Section ........................................................................................................... 17
2.2.1. Materials ....................................................................................................................... 17
2.2.2. Experimental procedure ................................................................................................ 19
2.3. Results and Discussion.................................................................................................... 21
2.3.1. Effect of IL Ions in Vanillin Partitioning ...................................................................... 21
2.3.2. Effect of Temperature in Vanillin Partitioning .............................................................. 24
2.3.3. Effect of Concentration in Vanillin Partitioning............................................................ 27
2.3.4. Density and Viscosity ................................................................................................... 28
2.4. Conclusions ......................................................................................................................... 36
3. Extraction of gallic acid in ATPS with ILs ............................................................................. 37
3.1. Gallic Acid ...................................................................................................................... 38
3.2. Experimental section ....................................................................................................... 40
3.2.1. Chemicals ................................................................................................................ 40
II
3.2.2. 3.2.2. Experimental procedure .................................................................................... 42
3.3. Results and Discussion.................................................................................................... 45
3.3.1. Phase Diagrams ....................................................................................................... 45
3.3.2. Effect of IL ions and pH in the acid gallic partitioning ........................................... 51
3.4. Conclusions ..................................................................................................................... 56
4. Future work............................................................................................................................. 57
5. References .............................................................................................................................. 59
List of Publications ......................................................................................................................... 67
Appendix A .................................................................................................................................... 69
A.1. Calibration curve for vanillin .............................................................................................. 70
A.2. Calibration curve for gallic acid .......................................................................................... 71
Appendix B ..................................................................................................................................... 72
Experimental data for the vanillin partition coefficients, density and viscosity .......................... 73
Appendix C ..................................................................................................................................... 79
van’t Hoff plots ........................................................................................................................... 80
Appendix D .................................................................................................................................... 81
Experimental binodal curve mass fraction compositions ............................................................ 82
Appendix E ..................................................................................................................................... 87
Experimental data of TL ............................................................................................................. 88
Appendix F ..................................................................................................................................... 89
Experimental data for the gallic acid partition coefficients ......................................................... 90
III
List of Tables
Table 1: Thermophysical properties of vanillin.[89, 92]
..................................................................... 13
Table 2: Standard molar thermodynamic functions of transfer of vanillin at 298.15 K. ................. 26
Table 3: Thermophysical properties of gallic acid.[92, 117]
................................................................ 38
Table 4: Initial weight fraction compositions for the determination of the phase diagrams and
indication of the possibility of existing liquid-liquid equilibrium. .................................................. 46
Table 5: Adjusted Parameters used to describe the experimental binodal data by Equation 5. ....... 49
Table 6: pH values as function the different systems performed. .................................................... 54
IV
List of Figures
Figure 1: Number of articles published per year concerning ILs. Data taken from IsiWeb of
Knowledge in 9th May, 2010. ............................................................................................................ 3
Figure 2: Cationic structures of nitrogen-based ILs. ......................................................................... 4
Figure 3: Structure of some phenolic compounds. ............................................................................ 8
Figure 4: Chemical structure of vanillin. ........................................................................................ 13
Figure 5: Production of vanillin from eugenol. ............................................................................... 14
Figure 6: Production of vanillin from sulfonated lignin fragments. ................................................ 14
Figure 7: Lignin precursors: alcohols p-coumaryl, coniferyl and sinapyl, respectively. ................. 15
Figure 8: Chemical structure of the studied ILs: (i) [C2mim]Cl; (ii) [C4mim]Cl; (iii) [C6mim]Cl;
(iv) [C7mim]Cl; (v) [C10mim]Cl; (vi) [amim]Cl; (vii) [C7H7mim]Cl; (viii) [OHC2mim]Cl; (ix)
[C4mim]Br; (x) [C4mim][CH3SO3]; (xi) [C4mim][CH3CO2]; (xii) [C4mim][CH3SO4]; (xiii)
[C4mim][CF3SO3]; (xiv) [C4mim][N(CN)2]. ................................................................................... 18
Figure 9: ATPS formed by IL + K3PO4 + H2O. ............................................................................... 19
Figure 10: Partition coefficients of vanillin in chloride-based ILs + K3PO4 ATPS at 298.15 K...... 22
Figure 11: Partition coefficients of vanillin in [C4mim]-based ILs + K3PO4 ATPS at 298.15 K. .... 23
Figure 12: Partition coefficients of vanillin in IL + K3PO4 ATPS as a function of temperature for
the ILs: [C4mim]Cl, [C4mim][CH3SO4], [C7H7mim]Cl and [amim]Cl. ........................................... 25
Figure 13: Partition coefficients of vanillin in IL + K3PO4 ATPS as a function of initial vanillin
concentration for the ILs: [C4mim]Cl, [C4mim][CH3SO4] and [C7H7mim]Cl. ................................ 27
Figure 14: Experimental viscosity (η) for the IL-rich phase (full symbols) and K3PO4-rich phase
(open symbols) for systems composed by chloride-based ILs + K3PO4 + H2O as a function of
temperature. .................................................................................................................................... 29
Figure 15: Experimental viscosity (η) for the IL-rich phase (full symbols) and K3PO4-rich phase
(open symbols) for systems composed by [C4mim]-based ILs + K3PO4 + H2O as a function of
temperature. .................................................................................................................................... 31
Figure 16: Experimental density (ρ) for the IL-rich phase (full symbols) and K3PO4-rich phase
(open symbols) for systems composed by chloride-based ILs + K3PO4 + H2O as a function of
temperature. .................................................................................................................................... 33
Figure 17: Experimental density (ρ) for the IL-rich phase (full symbols) and K3PO4-rich phase
(open symbols) for systems composed by [C4mim]-based ILs + K3PO4 + H2O as a function of
temperature. .................................................................................................................................... 34
V
Figure 18: Chemical structure of gallic acid. .................................................................................. 38
Figure 19: Chemical structure of the studied ILs: (i) [C7mim]Cl; (ii) [C8mim]Cl; (iii) [C4mim]Br;
(iv) [C4mim][CH3SO4]; (v) [C4mim][CF3SO3]; (vi) [C4mim][N(CN)2]; (vii) [C2mim][CF3SO3];
(viii) [C7H7mim] [C2H5SO4]; (ix) [C4mim][TOS]; (x) [C4mim] [C2H5SO4]; (xi) [C8py][N(CN)2];
(xii) [C7H7mim]Cl. ......................................................................................................................... 41
Figure 20: Experimental determination of the binodal curves for the aqueous systems IL-Na2SO4:
in the first picture it is shown a limpid and clear solution while in the second picture it denotes a
cloudy solution. .............................................................................................................................. 42
Figure 21: Ternary phase diagrams for all the ILs studied at 298 K and atmospheric pressure....... 47
Figure 22: Phase diagrams for the different ternary systems composed by IL+ Na2SO4+ H2O at 298
K and atmospheric pressure: ♦, experimental binodal data; □ ,TL data; ▬ fitting of experimental
data by the method proposed by Merchuck et al.[127]
...................................................................... 51
Figure 23: Partition coefficients of gallic acid for different ILs + Na2SO4 ATPS at 298.15 K. ....... 52
Figure 24: Partition coefficients of gallic acid in IL + different inorganic salt ATPS for the ILs:
[C2mim][CF3SO3], [C4mim][CF3SO3] and [C7mim]Cl. .................................................................. 53
Figure 25: Partition coefficients of gallic acid in IL + K2HPO4/KH2PO4 ATPS for
[C4mim][CF3SO3] at 25 wt % and 30 wt %. ................................................................................... 54
VI
Notation
List of symbols
Standard molar Gibbs energy of transfer
Standard molar enthalpy of transfer
Standard molar entropy of transfer
wt % Weight percentage
KGA Partition coefficient of gallic acid
KVan Partition coefficient of vanillin
R Universal gas constant
R2 Correlation coefficient
T Temperature
XB Inorganic salt weight percentage in the bottom
phase
XM Inorganic salt weight percentage in the
mixture
XT Inorganic salt weight percentage in the top
phase
YB Ionic liquid weight percentage in the bottom
phase
YM Ionic liquid weight percentage in the mixture
YT Ionic liquid weight percentage in the top
phase
α Ratio between the mass of the top phase and
the total mass of the mixture
ηB Viscosity of the bottom phase
ηT Viscosity of the top phase
ρB Density of the bottom phase
ρT Density of the top phase
σ Standard deviation
w1 Mass fraction composition of IL
w2 Mass fraction composition of Na2SO4
0
mtrG
0
mtrH
0
mtr S
VII
List of Abbreviations
ATPS Aqueous two-phase systems
BOD Biochemical oxygen demand
DNA Desoxyribonucleic acid
ILs Ionic liquids
LLE Liquid-liquid equilibrium
PEG Polyethylene glycol
PhCs Phenolic compounds
TL Tie-line
TLC Thin layer chromatography
TLL Tie-line length
UV Ultra-violet
VOCs Volatile organic compounds
[GA] Concentration of gallic acid
[GA]IL Concentration of gallic acid in the IL-rich aqueous phase
[GA]X Concentration of gallic acid in the inorganic salt-rich aqueous phase
[Van] Concentration of vanillin
[Van]IL Concentration of vanillin in the IL- rich aqueous phase
[Van]K3PO4 Concentration of vanillin in the K3PO4-rich aqueous phase
[C2mim]Cl 1-ethyl-3-methylimidazolium chloride
[C2mim][CH3SO4] 1-ethyl-3-methylimidazolium methylsulfate
[C2mim][CF3SO3] 1-ethyl-3-methyl-imidazolium trifluoromethanesulfonate
[C4mim]Cl 1-butyl-3-methylimidazolium chloride
[C4mim][CH3SO3] 1-butyl-3-methylimidazolium methanesulfonate
[C4mim][CF3SO3] 1-butyl-3-methyl-imidazolium trifluoromethanesulfonate
VIII
[C4mim][CH3SO4] 1-butyl-3-methylimidazolium methylsulfate
[C4mim][C2H5SO4] 1-butyl-3-methylimidazolium ethylsulfate
[C4mim][N(CN)2] 1-butyl-3-methylimidazolium dicyanamide
[C4mim][CH3CO2] 1-butyl-3-methylimidazolium acetate
[C4mim]Br 1-butyl-3-methylimidazolium bromide
[C4mim][HSO4] 1-butyl-3-methylimidazolium hydrogenosulfate
[C4mim][TOS] 1-butyl-3-methylimidazolium tosylate
[C6mim][Cl 1-hexyl-3-methylimidazolium chloride
[C7mim]Cl 1-heptyl-3-methylimidazolium chloride
[C8mim]Cl 1-octyl-3-methylimidazolium chloride
[C10mim]Cl 1-decyl-3-methylimidazolium chloride
[amim]Cl 1-allyl-3-methylimidazolium chloride
[amim][C2H5SO4] 1-allyl-3-methylimidazolium ethylsulfate
[OHC2mim]Cl 1-hydroxyethyl-3-methylimidazolium chloride
[C7H7mim]Cl 1-benzyl-3-methylimidazolium chloride
[C7H7mim][C2H5SO4] 1-benzyl-3-methylimidazolium ethylsulfate
[C4mpy]Cl 1-butyl-3-methylpyridinium chloride
[C4mpip]Cl 1-butyl-3-methylpiperidinium chloride
[C4mpyrr]Cl 1-butyl-3-methylpyrrolidinium chloride
[C8py][N(CN)2] 1-octylpyridinium dicyanamide
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
2
1.1. Scopes and Objectives
This work aims at studying the extraction of phenolic compounds (PhCs). Trying to develop more
benign extraction techniques than those used nowadays, in this work, a study was conducted using
aqueous two phase systems (ATPS) composed by ionic liquids (ILs) and typical inorganic salts.
The PhCs studied as partitioning molecules, and as examples of this general group, were vanillin
and gallic acid. PhCs present general attractive properties, such as their antioxidant, anti-
inflammatory, anti-microbial and anticarcinogenic capacities, among others.[1-4]
Due to these
interesting properties, PhCs have gained a special importance in food, wine, dietary and
pharmaceutical industries.[5-6]
The use of conventional volatile organic compounds (VOC’s) in extraction procedures brings up
some associated problems such as toxicity, volatility and flammability, and implies additional
environmental hazards. Moreover, some of these solvents can denaturate biomolecules and thus
influence their quality and purity.[7-8]
Currently ILs appear as a potential and alternative
replacement for VOC’s due to their negligible vapor pressures and intrinsic character of “designer
solvents”. The properties of ILs can be tunned by the proper choice of the cation and/or anion,
allowing the optimization of the physical and chemical characteristics that best adapt to a particular
process. The negligible vapor pressures makes also of ILs excellent candidates as alternatives to
VOC’s by decreasing atmospheric pollution concerns.[9-10]
Concerning the vanillin extraction, several parameters that could affect the solute extraction were
studied in ternary systems composed by imidazolium-based ILs, water and K3PO4, namely the
influence of the IL cation and anion nature, the temperature of extraction and the accessible
concentration of vanillin. Moreover, viscosities and densities of both the K3PO4-rich phase and the
IL-rich phase were measured in the temperature range from (298.15 to 318.15) K to evaluate the
additional advantages of using IL-based ATPS.
The extraction of gallic acid was studied in ternary systems composed by imidazolium-based ILs,
Na2SO4 and water. Due to the lack in literature of ternary phase diagrams containing ILs and the
salt Na2SO4, the individual phase diagrams, tie-lines and tie-line lengths were additionally
determined at 298 K. Both IL cation and anion nature were evaluated through the partitioning of
gallic acid. It is well known that the partitioning of an acidic solute is largely affected by the pH of
the system, and as a result, further partition coefficients were also determined in aqueous systems
formed by IL-K3PO4 and IL-K2HPO4/KH2PO4. The pH of each equilibrated phase was measured
and reported.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
3
1.2. Ionic Liquids
Ionic liquids (ILs) are compounds usually constituted by large and organic cations and organic or
inorganic anions with their melting points, by general definition, below 100 ºC.[11-13]
The low
symmetry, weak intermolecular interactions and a large distribution of charge in the ions is the
cause of the low melting points.[14-15]
ILs were first reported at the beginning of the 20th century by Paul Walden,
[16] when testing new
explosive compounds with the aim of replacing nitroglycerin. Walden synthesized ethylammonium
nitrate, [EtNH3][NO3] and found that it had a melting point around 13-14 ºC.[15]
The discovery of a
liquid salt did not received much attention at that time. In 1934, Charles Graenacher[17]
filled the
first patent for an industrial application for ILs regarding the preparation of cellulose solutions.
Later, during the World War II, ILs were again investigated and new patents were filled[18-19]
concerning the application of mixtures of aluminium chloride (III) and 1-ethylpyridinium bromide
to the electrodeposition of aluminium. Despite these findings, only in a recent past these
compounds have been extensively studied, and as can be seen in Figure 1, publications regarding
ILs exponentially increased from 1990 to 2009.
Figure 1: Number of articles published per year concerning ILs. Data taken from IsiWeb of
Knowledge in 9th May, 2010.
Among a large range of ILs that can be synthesized the most commonly studied are the nitrogen-
based ILs, namely pyrrolidinium-, imidazolium-, piperidinium-, pyridirium-, and ammonium-based
ILs, with their general cationic structures depicted in Figure 2. The cation can be highly complex
with different sizes for the alkyl side chains, different substitution positions and also additional
functional groups.[20]
0
1000
2000
3000
4000
5000
19
90
19
91
19
92
19
93
19
94
19
95
19
96
19
97
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00
20
01
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09
Nu
mb
er o
f art
icle
s p
ub
lish
ed
Year
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
4
Figure 2: Cationic structures of nitrogen-based ILs.
Due to the ionic nature of ILs, they present several physical and chemical advantages over
conventional and molecular organic solvents, namely negligible flammability and vapour pressure,
high solvation ability, high chemical stability, high selectivity, excellent microwave-absorbing
ability, and easiness in recovery and recycling.[5, 9, 21-24]
Apart from these advantages, many organic,
organometallic and inorganic compounds can be dissolved in ILs.[25]
ILs have also been
increasingly applied in catalysis[26]
, organic synthesis[21]
, chemical reactions[14]
, multiphase
bioprocess operations[27]
, electrochemistry[28]
, chromatographic separations[29]
, mass spectrometry
analysis[30]
, batteries and in fuel cells investigation[31]
and in the separation of biomolecules.[32]
Beyond these applications, ILs have also been used in liquid-liquid extractions of metal ions[33-34]
and organic compounds.[35-37]
Indeed, there are great interest on the application of ILs for the
removal of organic contaminants from aqueous solutions and in the use of ILs as solvents for
multiphase biotransformation reactions.[37-38]
It was already shown that most ILs do not inactivate enzymes ensuring their structural integrity and
enzymatic activity, and therefore ILs represent a good alternative to the usual solvents in
biocatalysis.[7, 22]
The ILs may allow an improved recovery of biomolecules when carrying liquid-
liquid extractions while reducing solvent emissions.
Since the ILs physicochemical properties are strongly dependent on the IL nature, the possibility of
changing their properties through the manipulation of the ions that compose them, represents an
important and supplementary advantage. This property - the “tunnability” - makes of ILs singular
compounds that can be designed with precise conditions for a particular process, as well as to
manipulate their extraction capabilities for specific biomolecules.[14, 23, 32]
In spite of the ILs potential environmental benefits as “green” replacements over conventional
volatile organic solvents, their toxicity must also be here discussed. Several studies[39-44]
were
conducted to evaluate the toxicity of ILs, combining different anions and cations, as well as
changing the alkyl group chain length and number of alkyl groups substituted at the cation ring.
These studies revealed that ILs toxicity is primordially determined by the cation nature and it is
directly correlated with the length of the side alkyl chain and number of alkyl groups. Commonly,
N+
R1
R2R3
R4
N+
R1
R2
N+
N
R2
R1
N+
R
N+
R1
R2
Imidazolium Pyridinium AmmoniumPyrrolidinium Piperidinium
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
5
the anion has a smaller influence than the cation, and generally short cation alkyl chains or
hydrophilic ILs present low or no toxicity.[41]
Usually, the ILs aqueous solubility decrease with the alkyl chain length increase, which for its turn
is positive because the more toxic ILs (higher alkyl chain lengths) are poorly water soluble at room
temperature, minimizing thus the environmental impact of ILs in aquatic streams.[41]
The choice of
the IL anion and cation determines the thermophysical properties of such fluids, and it was already
shown that the anion strongly influences their water miscibility.[41, 45]
The influence of the cation
alkyl chain length in the water solubility was also observed, but it can be considered minor when
compared with the anion influence.[45]
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
6
1.3. Extraction of Biomolecules Using Aqueous Two-Phase Systems
(ATPS)
There are two main processes commonly used to extract (bio)compounds from a liquid phase to
another: liquid-liquid extraction using immiscible solvents and aqueous two-phase systems
(ATPS). These are formed when two mutually incompatible, though both miscible with water, are
dissolved in water.
Liquid-liquid extraction processes are used for the purification of biomolecules due to their high
effectiveness, high yield, improved purity degree, proper selectivity, technological simplicity and
low cost, and also to a good combination between the recovery and purification steps.[7, 32, 46]
The
extraction of biomolecules is usually carried out using organic solvents because of their
immiscibility with aqueous media.[47]
The most common organic solvents used present some
disadvantages, such as high volatility and toxicity and the possibility of denaturating biomolecules,
which in turn may influence the quality and purity of these to be recovered.[7]
Aiming at avoiding the use of organic solvents as the extractive phase, several attempts have been
carried out employing ATPS. Separation of biological molecules and particles using ATPS dates to
1958 and were introduced by P. A. Albertsson.[48]
ATPS consist in two aqueous-rich phases
containing polymer/polymer, polymer/salt or salt/salt combinations.[49]
The basis of separation of
(bio)molecules in a two-phase system results from their equilibration and selective distribution
between the two liquid aqueous phases.[50]
For a while ATPS were used only in laboratories but by their simplicity, biocompatibility, and ease
of scale-up operations, their use has expanded to large-scale (bio)separations.[50]
Furthermore, it
was already shown that it is possible to recover and separate a wide range of biomaterials using
ATPS, such as plant and animal cells, microorganisms, proteins, among others.[48, 51-54]
From the
industrial point view, ATPS extraction represent no major problems because engineering and
existing equipments are easily adapted to the requirements. Indeed, a number of proteins are
purified by this process at an industrial level.[50]
Common ATPS are usually formed by polyethylene glycol (PEG) because it easily forms a
biphasic system with inorganic salts and neutral polymers in aqueous solutions. Some hydrophilic
polymers are immiscible among them. The separation of phases coexists in the equilibrium
between them, and with each phase containing predominantly water and one of the polymers. The
water mixed with polymers acts as the major solvent and can establish non-covalent bonds with
them. These interactions increase with the molecular weight of the polymers, and phase separation
can occurs at very low polymer concentrations due to the loss of entropy in the demixing
process.[55-56]
On the other hand, the presence of an inorganic salt in critical concentrations in a
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
7
single polymer-water system can also lead to the formation of two distinct aqueous phases, where
usually the lower phase is rich in the inorganic salt, while the upper phase is rich in polymer. The
phase separation certainly depends on the type of inorganic salt and respective concentration (for
example, K2HPO4, K3PO4, K2CO3, KOH, Na2HPO4 and Na2SO4 are typical inorganic salts
employed). [53, 56]
In general, both phases are composed by approximately 70-90 % of water, which
means that biomolecules are not easily denatured, constituting therefore an important advantage
when the goal is to extract proteins and/or enzymes.[50, 57]
It should be pointed out that the
partitioning of a biomolecule in ATPS can vary depending on the biomolecule size, surface
properties, molecular weight, temperature, pH, net charge, among others.[58]
In both type of ATPS
mentioned, the interactions between a biomolecule and the distinct phases could involve hydrogen
bonds, van der Waals-, dispersive-, and electrostatic-interactions, as well as steric-, and
conformational effects.[32, 53]
To improve the extraction efficiency, the replacement of ordinary organic solvents by ILs has been
suggested as a promising alternative. Organic solvents may be substituted directly using
hydrophobic ILs as a second immiscible liquid phase with aqueous media[59]
or by the use of ATPS
incorporating hydrophilic ILs.[8, 32, 49, 60-61]
The second approach is more used because ATPS
containing hydrophilic ILs have shown to be more effective in extracting biomolecules.[32, 59]
Gutowski et al.[60]
were the first to show that aqueous solutions of imidazolium-based ILs can form
ATPS in presence of aqueous solutions of certain inorganic salts, such as K3PO4. Since then, the
equilibrium properties of systems comprising ILs, for the development of specific extraction and
isolation procedures, have been investigated.[41-42, 59, 61-65]
It was already shown that ATPS formed
by K3PO4 and ILs are extremely advantageous for the partitioning of several biomolecules yielding
larger partition coefficients than those conventionally obtained with polymers-inorganic salts or
polymers-polysaccharides ATPS.[32, 49, 61]
The extractive potential of biomolecules using IL-based
ATPS was previously studied for distinct compounds, such as testosterone and epitestosterone,
alkaloids, antibiotics, bovine serum albumin, penicillin G, L-tryptophan and food coulorants
[12, 32,
49, 57, 61, 66-68]. These results clearly indicated an attractive prospective of IL-based ATPS for
biomolecules separation and purification.[32, 49]
The effect of inorganic salts on ATPS formation has
already been widely studied [12, 57, 60, 69-70]
while demonstrating to follow the Hofmeister series[71]
(classification of ions based on their salting-out/-in ability). Concerning the ILs in a previous
work[32]
it was shown that the ability of an IL to form ATPS is related with the decrease in the
hydrogen bond accepting strength or the increase in the hydrogen bond basicity of the IL anion.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
8
1.4. Phenolic compounds (PhCs)
Phenolic compounds, PhCs, are compounds constituted by at least one phenol group, i.e., a benzene
ring with a hydroxyl group (-OH). Examples of these compounds are vanillic, gallic,
protocatechuic, ellagic, syringic, caffeic, gentisic and ferulic acids, and quercetin, vanillin and
resveratrol, among others. These compounds may be present in natural sources such as wood, fruits
and vegetables. [1-2, 6, 72-73]
Some of these examples are depicted in Figure 3.
Figure 3: Structure of some phenolic compounds.
PhCs present redox properties that allow them to act as hydrogen donors and singlet oxygen
quenchers. Since redox reactions involve the transport of electrons, PhCs present antimicrobial
action.[73]
The oxidation of unsaturated fats by free radicals, such as reactive oxygen species, can
cause biological changes inside the human body, namely heart disease, arthritis, cellular
degeneration related to aging, changes in DNA and even cancer. The antioxidant action of PhCs
protects cells by neutralizing free radicals as oxygen free radicals and by decomposing
peroxides.[74-75]
Indeed, in literature,[76-77]
it was already shown the ability of PhCs to protect cells
from oxidative stress.
PhCs have particular characteristics of utmost importance. Besides their high antioxidant capacity,
they are also phytotoxic and toxic to bacteria, and used thus in biological wastewater treatments,
and present toxicity against human promyelocytic leukemia cells.[1-2, 4]
A previous study[73]
revealed
that the antimicrobial action of PhCs is independent of pH. PhCs can also act as prooxidants rather
than antioxidants, depending on their concentration and free radical source. Therefore, the
cytotoxicity of PhCs is linked to their prooxidant properties.[4]
The presence of PhCs in plants represents the major source of these compounds from biomass, and
some benefits, besides the antioxidant activity, are attributed to them, such as the ability of
lowering cholesterol, depression of hypertension, protection against cardiovascular disease, among
others.[78]
Due to these properties PhCs have been object of special interest in food, wine, dietary and
pharmaceutical industries.[5-6]
Gallic acid Ellagic acid Quercetin Vanillin
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
9
Wood is composed by cellulose, hemicelluloses, lignin and extractables. It is a renewable raw
material resource (biomass) and can be used for the production of new materials, energy and
several chemical products. Nowadays, wood plays a key role in the production of various
commodities and have progressively replaced some products from the petrochemical industry. The
PhCs present in the extractables play an important role in the protection of wood against
microbiological harm or insect attacks. Moreover, PhCs also contribute to the natural color of
wood. Main products such as gallic and ellagic acid, and sugars can be produced from hydrolysable
tannins.[79]
Besides the presence of PhCs in wood and plants, they can also be found in many residues from the
industrial or agricultural activities, such as wastewaters from olive mills. These residues may
contain 4-16 % of organic matter, and from which PhCs represent 2-15 %.[1]
To decrease the load of organic compounds in effluents, and at the same time add value to the
process, there is a growing interest in their extraction both from natural sources and industrial or
agricultural waste.[1]
Concerning this subject, there are some published studies regarding the
extraction of phenolic compounds.[5, 72, 80-90]
It is important to note that depending on the phenolic
compound to be extracted, the improved technique or optimum solvent may be quite different.
Brudi et al.[81]
studied the partition coefficients of some aromatic organic substances, such as
vanillin and phenol, in a two-phase mixture of water and carbon dioxide. The partition coefficients
were determined by the ratio between the mole fraction solubilities between the carbon dioxide and
the water phases. The experiments were carried out at high temperature and pressure conditions
using carbon dioxide as a supercritical fluid.[81]
The attained partition coefficients for phenol varied
between 0.2 and 1.5.[81]
On the other hand, the partition coefficients of vanillin ranged between 0.2
and 3. An increase in temperature was found to decrease the partition coefficients.[81]
Adrian et
al.[80]
investigated the partition coefficients of vanillin and caffeine in an high pressure multiphase
equilibrium system. The system was composed by carbon dioxide, water, propanol and a small
concentration of the partitioning solute.[80]
The authors reported that vanillin preferentially migrates
for the organic phase (KVan ≥ 1) and that an increase in pressure increases the partition coefficients
of both solutes.[80]
Following the approach of high pressure systems, also Kim et al.[87]
studied the
extraction of phenolic compounds (p-hydroxybenzaldehyde, vanillic acid, syringic acid, vanillin,
acetovanillone, and feruric acid) by pressurized low-polarity water (PLPW).
Regarding organic-aqueous two phases systems and to evaluate the PhCs environmental impact,
Noubigh et al.[72]
recently determined the partition coefficients between octanol and water (KOW)
for PhCs such as protocatechuic acid, vanillic acid, and vanillin, as a function of temperature, using
the slow-stirring method. The KOW values of protocatechuic acid, vanillic acid, and vanillin varied
between 6.03 and 4.07, 28.84 and 26.18, and 17.78 and 9.77, respectively. In addition, Tarabanko
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
10
et al.[89]
used octylamine-based systems for the extraction of vanillin and found that the distribution
ratios increased with the octylamine concentration and in a pH range between 8 and 10. Octylamine
provided high distribution ratios, and when using moderate concentrations of octylamine and high
pH values, high extraction selectivities could be achieved.[89]
Moreover it was also found that the
extraction of vanillin decreases with the use of additional anti-solvents, such as hexane, toluene,
benzene, dichloroethane and chloroform.[89]
Horax et al.[85]
extracted phenolics compounds from pericarp and seeds of bitter melons with
ethanol and water systems. The extraction efficiency of PhCs was found to change with the ethanol
concentration.[85]
Moreover, Trabelsi et al.[90]
investigated several solvents and/or mixtures of
solvents on the phenolic compounds extraction efficiencies. The extraction ability of solvents for
the extraction of phenolic compounds from leaves decreased in following order: methanol >
ethanol/water > acetone/water > methanol/water > methanol/HCl ≈ acetone >>
methanol/ethanol/acetone ≈ water >hexane> ethanol.[90]
In this study it was found that more polar
solvents are more efficient in the extraction of PhCs compounds.[90]
These results are in agreement
with others in literature[84, 88]
, where it was shown that methanol is an improved extractive phase
compared to ethanol. In addition, others authors[83]
investigated the extraction of PhCs from
different natural sources using water and organic solvents mixtures, namely acetone/water/acetic
acid and ethyl acetate/methanol/water. The authors concluded that the solvent employed has a
substantial influence through the extraction of PhCs compounds from Quercus coccifera L. and
Juniperus phoenicea L.[83]
Following the same type of studies, Khokhar[91]
postulated that water is
the best extracting solvent for PhCs from tea catechins when compared with solutions of 80 % (v/v)
of methanol or 70 % (v/v) of ethanol.
Jadhav et al.[86]
performed a study with the goal of comparing conventional soxhlet and ultrasound-
assisted extraction of vanillin from vanilla pods. Also the solvent influence was assessed and it was
found that the extraction of vanillin decreases in the following rank: ethanol > methanol > acetone
> acetonitrile > chloroform > hexane.[86]
Again the results obtained revealed that polar solvents are
more indicated for the extraction of PhCs.[86]
Comparing both techniques, the ultrasound-assisted
extraction was found to be more efficient.[86]
In addition to the extractions with pure solvents,
mixtures of water–ethanol were also studied.[86]
The results indicated that the extraction of vanillin
from cured vanilla beans was greatly influenced by the mixture composition, where a maximum in
extraction was achieved with a mixture composed by 50 % (v/v) of ethanol.[86]
Hasmann et al.[82]
have used aqueous two-phase systems composed by two thermoseparating
copolymers (ethylene oxide and propylene oxide) to remove phenolic compounds from
hemicellullosic hydrolysate (from rice straw). In this study[82]
several parameters were evaluated,
such as the copolymer molecular weight and the mass fraction compositions of the system. The
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
11
thermoseparation was carried out resulting in the formation of a two-phases system consisting of a
top copolymer-rich phase and a bottom hydrolysate-rich phase.[82]
The extraction efficiencies of
PhCs varied from 6 % to 80 % depending on the copolymer employed. Curiously, in a system
containing 50 wt % of the copolymer with the higher molecular mass, it was not observed the
extraction of PhCs due to an high increase in the viscosity of the system. Also, higher mass fraction
contents of copolymer (more than 35 wt %) were found unfavorable for PhCs extraction in the
studied ATPS.[82]
Moreover, the use of ILs for the extraction of PhCs from medicinal plants was
already studied by Du et al.[5]
using microwave-assisted extraction (MAE). When comparing the
extraction efficiencies between ILs aqueous solutions and pure water, the extraction yields of
polyphenolic compounds are greatly improved by the addition of ILs.[5]
Indeed, no major
differences were found in the extraction efficiencies between methanol and ILs aqueous solutions.
In the optimized conditions, the extraction yields of the polyphenolic compounds varied between
79.5 % and 93.8 % in an one-step extraction. Using different ILs it was also concluded that cations,
and especially the anions of ILs, affect the extraction efficiencies of polyphenolic compounds.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
12
2. Extraction of vanillin in
ATPS with ILs
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
13
2.1. Vanillin
Vanillin, 3-methoxy-4-hydroxybenzaldehyde, presents the chemical formula CH3O(OH)C6H3CHO,
and its appearance is a white crystalline powder with an intense and pleasant odor. This
biomolecule is relatively soluble in chloroform, ether and water at room temperature. The
properties of vanillin are reported in Table 1.
Table 1: Thermophysical properties of vanillin.[89, 92]
Molar mass
(g·mol-1
)
Density
(g·cm-3
)
Melting
point (K)
Boiling
point (K)
Solubility in
water (g·dm-3
) pKa
152.15 2.056 at 298 K 353-354 558 10 at 298 K 8.2 at 298 K
Vanillin, Figure 4, is a biomolecule relevant for several purposes, whose recovery and purification
by cost-effective and environmentally-safe processes is still of major interest. In 1858, Gobley[93-96]
isolated all the components of vanilla detecting that vanillin is
the major compound and that confers to it the well known
organoleptic properties. Therefore, vanillin is one of the mostly
appreciated fragrant substances aiming at creating artificial
aromas in a wide range of commercial products. Vanillin or
vanilla are currently used in foods, beverages, livestock fodder
and pharmaceutical products, as well as in the fragrance industry
with the aim of creating perfumes, or masking unpleasant odors,
and cleaning products.[97]
Beyond these aroma and fragrance
applications, vanillin is also used as a chemical intermediate in the production of pharmaceuticals
and fine chemicals for use in biocides (due to its phenolic character) and specialty chemicals in
technical applications, such as TLC (thin layer chromatography).[72, 98]
Due to the scarcity of vanillin in natural sources, the synthesis and complex routes for the
extraction of this compound were explored.[98]
Initially, between 1874 and 1920[98-99]
, the synthesis
of vanillin resulted from the reaction involving eugenol compounds taken from oil of cloves. The
reaction[98]
that occurs to generate vanillin from eugenol is given by:
Figure 4: Chemical
structure of vanillin.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
14
Figure 5: Production of vanillin from eugenol.
Other later alternatives relayed on the synthesis of vanillin from guaiacol and/or lignin.[98]
In the
paper production, lignin is withdrawn and so, it is a byproduct of the paper industry. Lignin is a
component of wood and typically represents 30 % of its constitution.[79]
The first signs of
producing vanillin from lignin-containing wastes date to 1875 through an anonymous report.[100]
Years later, Grafe[101]
confirmed such point through the pyrolysis of dried waste sulfite liquor.[98]
After this report,[100]
this method of synthesizing vanillin was further explored by several
researchers. Howard,[102]
and other researchers from the United States, patented some methods in
order to concentrate the lignin present in the pulping waste. Tomlinson and Hibbert[103]
revealed
that the yield of vanillin obtained depends on the source of the liquor and on the degree of lignin
sulfonation. The authors[103]
were able to demonstrate that vanillin can be recovered up to 2.6 g·dm-
3 (5.9 % of the lignin content) from waste sulfite liquor. Later, Freudenberg
[104] proved that the
oxidation of lignin from softwoods results in the formation of vanillin, and yielding 10 % using air,
or 20–30 % using nitrobenzene as oxidant.[79]
Despite the higher yield that was obtained with the
use of nitrobenzene, these findings were unappealing from the commercial point of view because
of the need to deal with the co-produced nitrobenzene.[98]
To reaction for synthesizing vanillin from the sulfonated lignin fragment from the purified liqueurs
can be described by:[98]
Figure 6: Production of vanillin from sulfonated lignin fragments.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
15
In 1936, in North America, the production of vanillin from the lignin-containing waste liquor
(“brown liquor”), acquired at the sulfite process, was largely explored using the methodology
proposed by Howard.[105]
Consequently, in the following years, two production-scale plants were
built in Ontario, Canada.[98]
In 1937 it was built the Howard Smith Paper Mills Ltd., in Cornwall,
Ontario, based on the study developed the McGill University[106]
. In 1945, in Thorold, Ontario, it
has been created another vanillin-from-lignin production unit through the study conducted by
Ontario Pulp and Paper in the 1940s.[98]
By this implementation at the industrial scale, vanillin
became more abundant and less expensive. Indeed, in 1981, a single plant of pulp in Ontario
produced 60 % of worldwide vanillin consumed. In this plant, the synthesis of vanillin results from
an initial fermentation that uses some fermentable sugars usually present in spent acid sulfite
pulping. During the fermentation black liquor alkaline oxidation, the oxidation product stream is
extracted with toluene and further back-extracted with aqueous sodium hydroxide. These steps
generate an aqueous solution of crude sodium vanillate which is further purified via the carbonyl
sulfite by the addition of aqueous sulfur dioxide. The soluble additional compounds are separated
from acetovanillone and other insoluble phenolic impurities by filtration. In order to produce a
product for typical use in the food industry it is also necessary to reprecipitate vanillin from this
aqueous solution.[98]
The vanillin synthesized through the appropriate treatment of sulfite liquor waste is produced from
guaiacyl units of lignin, which is sulfonated and solubilized in the process of transforming wood
into chemical pulp to produce paper. To have a high yield of vanillin, the wood used in the
chemical process must be of the softwood type (this type of wood is the main precursor of coniferyl
alcohol and thus has more guaiacyl units to produce vanillin - called the G lignin (Guaiacyl
lignin)). In hardwood the precursor coniferyl and sinapyl alcohol create the GS lignin (Syringyl-
Guaiacyl lignin), whose percentage of units of guaiacyl is very low when compared to
softwoods.[79, 98]
The lignin precursors are shown in Figure 7.
Figure 7: Lignin precursors: alcohols p-coumaryl, coniferyl and sinapyl, respectively.
CH2OH
CH
CH
OH
OCH3
CH2OH
CH
CH
OH
H3CO OCH3
CH2OH
CH
CH
OH
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
16
After alkaline oxidation, in liquor pulping, vanillin is majorly obtained from softwoods (25 %),
unlike to what happens for hardwoods, where the alkaline oxidation leads mainly to the production
of syringic aldehyde and to a lower percentage of vanillin.
Although the previous approaches have indicated the synthesis of vanillin from the purified liquor
(without sugars) from the acid sulfite method, it should be pointed out that it is also possible to
obtain vanillin performing an extraction with an alcohol from the pre-concentrated spent black
liquor of kraft (sodium hydroxide and sodium sulfide based).[107]
As main disadvantage, lignin-based vanilla is believed to have a richer flavor than the vanilla-based
oil. This fact results from the presence of acetovanillone in the lignin-derived products. This
compound is an impurity that is not present in vanillin synthesized via guaiacol.[97]
Initially, the development of methods for the synthesis of vanillin from waste sulfite liquor
presented main advantages due to the low contents of solubilized sugars and lignin content
(biochemical oxygen demand or BOD properties) of the spent liquor. Nevertheless, after the
vanillin recovery process there is the further need of removing ≈ 160 kg of "liquid caustic" per each
kilogram of vanillin produced, making therefore the process not sustainable as it was seemed a
priori. As a result, residual vanillin-from-lignin plants in Canada and United States, such as the
Thorold plant, were closed.[98]
However, this practice is still under operation in a number of small
plants.[98]
On the other hand, vanillin from guaiacol-based routes is gaining an increasing interest
albeit guaicol is not a renewable source.
The development of new techniques for vanillin separation and purification from several matrices,
while maintaining their functional characteristics unchanged, is still ongoing. New and more
benign approaches for the selective extraction of vanillin from lignin-containing waste liquor or
agricultural or industrial residues should be explored. It should be pointed out that the results
presented hereinafter represent a proof of principle of the potential use of IL-based ATPS to carry
out such extractions.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
17
2.2. Experimental Section
Attempting at understanding the molecular mechanisms behind the partition of biomolecules in
ATPS containing ILs, and to identify the improved ILs for the extraction of PhCs, in this work an
extensive study was conducted using vanillin, a phenolic aldehyde, as the partitioning molecule.
For that purpose, several extraction parameters were studied in ternary systems composed by
imidazolium-based ILs, water and K3PO4, namely the influence of the IL cation and anion, the
temperature of extraction and the concentration of vanillin. Moreover, viscosities and densities of
both the inorganic salt-rich phase and the IL-rich phase were measured in the temperature range
from (298.15 to 318.15) K to evaluate the advantage of using IL-based ATPS.
In this study, the inorganic salt employed for the extractions was K3PO4 that confers an alkaline pH
to the aqueous system. In the industrial process, based on the lignosulfonate oxidation to originate
vanillin, the pH of the medium is also highly alkaline.[89]
2.2.1. Materials
Since it is highly difficult to perform measurements through all the possible combinations between
cations and anions in ILs it is thus crucial to perform measurements on selective systems aiming at
providing results that can be further used as optimized extractions procedures. Therefore, this work
evaluated the extraction ability of several IL-based ATPS for vanillin. All ILs studied are based on
the imidazolium cation: 1-ethyl-3-methylimidazolium chloride, [C2mim]Cl; 1-butyl-3-
methylimidazolium chloride, [C4mim]Cl; 1-hexyl-3-methylimidazolium chloride, [C6mim]Cl; 1-
heptyl-3-methylimidazolium chloride, [C7mim]Cl; 1-decyl-3-methylimidazolium chloride,
[C10mim]Cl; 1-allyl-3-methylimidazolium chloride, [amim]Cl; 1-benzyl-3-methylimidazolium
chloride, [C7H7mim]Cl; 1-hydroxyethyl-3-methylimidazolium chloride, [OHC2mim]Cl; 1-butyl-3-
methylimidazolium bromide, [C4mim]Br; 1-butyl-3-methylimidazolium methanesulfonate,
[C4mim][CH3SO3]; 1-butyl-3-methylimidazolium acetate, [C4mim][CH3CO2]; 1-butyl-3-
methylimidazolium methylsulfate [C4mim][CH3SO4]; 1-butyl-3-methyl-imidazolium
trifluoromethanesulfonate, [C4mim][CF3SO3]; 1-butyl-3-methylimidazolium dicyanamide,
[C4mim][N(CN)2]. The ILs were supplied by Iolitec. To reduce the water and volatile compounds
content to negligible values, ILs individual samples were dried under constant agitation at vacuum
and moderate temperature (≈ 343 K) for a minimum of 24 hours. After this procedure, the purity of
each IL was further checked by 1H,
13C and
19F NMR spectra and found to be > 99 wt % for all
samples. The inorganic salt K3PO4 was from Sigma with a purity level > 98 wt %. Vanillin, > 99 wt
% pure, was from Aldrich. The molecular structures of the studied ILs are depicted in Figure 8. The
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
18
water employed was double distilled, passed across a reverse osmosis system and further treated
with a Milli-Q plus 185 water purification apparatus.
Figure 8: Chemical structure of the studied ILs: (i) [C2mim]Cl; (ii) [C4mim]Cl; (iii) [C6mim]Cl;
(iv) [C7mim]Cl; (v) [C10mim]Cl; (vi) [amim]Cl; (vii) [C7H7mim]Cl; (viii) [OHC2mim]Cl; (ix)
[C4mim]Br; (x) [C4mim][CH3SO3]; (xi) [C4mim][CH3CO2]; (xii) [C4mim][CH3SO4]; (xiii)
[C4mim][CF3SO3]; (xiv) [C4mim][N(CN)2].
N+
N
Cl-
N+
NCl-
N+
NCl-
N+
N
Cl-
N+
NCl-
iii
iii
iv v
N+
NCl-
N+
NCl-
N+
N
OHCl-
vi viiviii
N+
N
Br-
ixS
O
O
-O
O
O-
N+
N
N+
N
x xi
N+
N
S
O
O
-O
O
S OO
O-
F
F
F
N+
N
N
N N
N+
Nxii
xiii xiv
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
19
2.2.2. Experimental procedure
2.2.2.1. Vanillin Partitioning
A ternary mixture was prepared within the biphasic region containing 15 wt % of K3PO4, 60 wt %
of an aqueous solution of vanillin and 25 wt % of all above mentioned ILs. Only for [OHC2mim]Cl
a different composition (15 wt % of K3PO4, 40 wt % of IL and 45 wt % of the aqueous solution of
vanillin) was used due to the smaller two-phase region obtained within
this IL. The ternary mixtures compositions were chosen based on the
phase diagrams of each IL-K3PO4 system reported in previous works.[32,
49] For all the ternary mixtures evaluated, and at the compositions used,
the top layer is the IL-rich phase while the bottom phase is the K3PO4-rich
phase, and as can be seen in Figure 9.
The ternary compositions were prepared by weight with an uncertainty of
± 10-5
g. The vanillin content influence was studied using different
concentrations of the compound at the aqueous phase composition (0.5
g·dm-3
,
1.0 g·dm-3
, 2.5 g·dm-3
, 5.0 g·dm-3
and 7.5 g·dm-3
which
correspond to 3.3 × 10-3
mol·dm-3
, 6.6 × 10-3
mol·dm-3
, 1.6 × 10-2
mol·dm-
3, 3.3 × 10
-2 mol·dm
-3 and 4.9 × 10
-2 mol·dm
-3, respectively).
Each mixture (IL, K3PO4 and aqueous solution of vanillin) was vigorously stirred and allowed to
reach equilibrium by the separation of both phases for 12 h and at the temperature of interest using
small ampoules (10 cm3) especially built for such extraction procedures. A preliminary study
showed that the equilibration of vanillin was attained after a period of 12 h. The time required to
establish the equilibrium of vanillin was experimentally determined by measuring the concentration
of vanillin in each phase at different times until reproducible data were obtained. The temperatures
evaluated were 288.15 K, 298.15 K, 308.15 K, 318.15 K and 328.15 K within an uncertainty of ±
0.01 K, and attained using an air bath equipped with a Pt 100 probe and a temperature controller or
making use of a refrigerated water bath, Julabo F34. After a careful separation of both phases, the
amount of vanillin at each aqueous phase was quantified through UV-spectroscopy, using a
SHIMADZU UV-1700, Pharma-Spec Spectrometer, at wavelength of 280 nm. Calibration curves
were properly established and are reported in Appendix A. At least three individual samples of
each phase were quantified in order to determine the vanillin partition coefficients and the
respective standard deviations. Possible interferences of both K3PO4 and all ILs with the analytical
method were investigated and found to be not significant at the working conditions used.
The partition coefficients of vanillin, KVan, were determined as the ratio of the concentration of
vanillin in the IL and in the inorganic salt (K3PO4) aqueous-rich phases, accordingly to:
Figure 9: ATPS
formed by IL +
K3PO4 + H2O.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
20
43POK
ILVan
Van
VanK (1)
where [Van]IL and [Van]K3PO4 are the concentration of vanillin in the IL and in the inorganic salt
aqueous rich phases, respectively.
2.2.2.2. Density and Viscosity
Density and viscosity of the phases formed during the vanillin extraction were measured using an
automated SVM 3000 Anton Paar rotational Stabinger viscometer-densimeter in the temperature
range from (298.15 to 318.15) K, within ± 0.02 K. The dynamic viscosity has a relative uncertainty
within 0.35 %, while the absolute uncertainty in density is ± 5 × 10-4
g·cm-3
.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
21
2.3. Results and Discussion
The extraction of a molecule with ATPS strongly depends on the ability to manipulate the
physical/chemical properties of the phases aiming at obtaining high partition coefficients and
specific selectivity for the biomolecules of interest. Several advances can be used to control the
molecules partitioning, such as the chemical nature of the system, temperature of equilibrium,
system composition and inclusion of antisolvents, co-solvents or amphiphilic structures. Three
process variables were evaluated in this work: the IL cation and anion nature, the temperature of
extraction, and the initial concentration of vanillin. To optimize the ILs to be used and operating
conditions for the vanillin extraction, it is of high importance to understand the physicochemical
issues that rule the biomolecule partitioning between the two equilibrated aqueous-rich phases. The
values of the partition coefficients between the two phases result from a complex balance between
IL-vanillin, K3PO4-vanillin and water-vanillin interactions and are determined by the relative
strengths of the interactions of the biomolecule with each of the compounds present on the system.
Taking into account the vanillin and ILs molecular structures depicted in Figure 4 and Figure 8,
these interactions may result from dispersive forces, electrostatic interactions, hydrogen-bonding,
steric and conformational effects, molecular size and ··· stacking between.[32, 53, 108]
The
partitioning of vanillin into one phase requires the disruption of interactions between its
components to create a cavity where the solute can be accommodated. It is thus expected that
vanillin will partition to a phase where less energy is required to create a cavity and that the new
interaction formed between vanillin and its solvation neighbors are indeed more favorable. For all
the studied systems it was observed that vanillin preferentially migrates for the IL-rich phase (KVan
> 1) resulting from the favorable interactions between vanillin and imidazolium-based ILs, and
from the lower energy required to create a cavity in the IL-rich phase due to the lower surface
tension of this phase.[109]
The mass fraction compositions used for the determination of each
partition coefficient, as well as the partition coefficients values and respective standard deviations,
are presented in Appendix B.
2.3.1. Effect of IL Ions in Vanillin Partitioning
In order to evaluate the IL ions influence in the extraction of vanillin several combinations were
performed. For the cation influence study, the chloride anion was kept, while combined with the
following cations: [C2mim]+, [C4mim]
+, [C6mim]
+, [C7mim]
+, [C10mim]
+, [amim]
+, [OHC2mim]
+
and [C7H7mim]+. The selected ILs allow the study of the alkyl side chain length effect, as well as
the study of additional functional groups. The influence of the IL anion was evaluated through the
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
22
use of the [C4mim]+ cation combined with the following anions: Cl
-, Br
-, [CH3CO2]
-, [CH3SO3]
-,
[CF3SO3]-, [CH3SO4]
- and [N(CN)2]
-. All of these studies were performed at 298.15 K.
The partition coefficients measured at 298.15 K are presented in Figures 10 and 11 and show that
KVan ranges between 2.72 and 49.59 (at approximately the same mass fraction compositions of IL
and inorganic salt).
Figure 10: Partition coefficients of vanillin in chloride-based ILs + K3PO4 ATPS at 298.15 K.
Regarding the IL cation influence, the ability of ILs to extract vanillin follows the order:
[C6mim]Cl > [C4mim]Cl > [C7H7mim]Cl > [C7mim]Cl > [C2mim]Cl ≈ [amim]Cl > [OHC2mim]Cl
>> [C10mim]Cl. Increasing the alkyl side chain of the imidazolium cation there is an increase on
the vanillin partition coefficients, reaching a maximum with [C6mim]Cl, followed by a decrease
until [C10mim]Cl. Indeed, the lowest partition coefficient of vanillin was observed for the IL with
the longest cation alkyl chain length, [C10mim]Cl. Increasing the size of the alkyl side chain
increases the IL free volume, while decreasing the surface tension of the system,[109]
and thus
decreasing the energy of cavity formation to accommodate a vanillin molecule. However, the
increase of the alkyl side chain length generates the formation of ILs aggregates in aqueous
solutions increasing thus the ILs affinity for water.[110]
This increase of the alkyl side chain length
also promotes a steric hindrance effect that leads to a decrease on the coloumbic and polar
interactions, while increasing the dispersion interactions, between the IL ions. [42, 111]
Therefore,
these two contributions will act in different directions leading to the observed maximum value on
the partition coefficients at about [C6mim]Cl.
36.49
44.98
49.59
42.39
2.72
44.19
36.45
22.95
0
10
20
30
40
50
60
KVan
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
23
The presence of a double bound, an aromatic or an hydroxyl group at the imidazolium alkyl chain
increases the IL hydrophilicity or the IL affinity for water.[49]
Nevertheless, the partition
coefficients of vanillin were not significantly enhanced using [C7H7mim]Cl, [amim]Cl or
[OHC2mim]Cl when compared with the values obtained for [C7mim]Cl, [C4mim]Cl and
[C2mim]Cl. Although differences are observed with the IL cation, especially with the alkyl side
chain length, the addition of functional groups does not have a particular effect on the partitioning
of vanillin. Indeed, only for [C7H7mim]Cl the partition coefficient increases slightly compared to
[C7mim]Cl, although they are not statistically different taking into account the associated standard
deviations. The results indicate that an increase in the IL cation hydrophilic character by the
inclusion of additional functional groups does not improves the vanillin extraction. It seems thus
that the cation-anion interaction strengths are the major forces driving the partitioning of vanillin.
Weaker coulombic forces allow an easy access of vanillin to interact both with the IL cation and
anion.
Regarding the IL anions effect on the vanillin extraction, shown in Figure 11, the following rank
was observed: [C4mim]Cl > [C4mim][N(CN)2] > [C4mim]Br > [C4mim][CH3SO4] >
[C4mim][CF3SO3] > [C4mim][CH3SO3] ≈ [C4mim][CH3CO2].
Figure 11: Partition coefficients of vanillin in [C4mim]-based ILs + K3PO4 ATPS at 298.15 K.
Vanillin partitions preferentially for IL-rich phases composed by halogenated ions, such as Cl- or
Br-, or for ILs comprising anions with a more hydrophobic character and a higher hydrogen
44.98
31.87
25.6622.74
9.756.94 6.64
0
10
20
30
40
50
KVan
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
24
bonding accepting character, such as [N(CN)2]-. In addition, the fluorination of [C4mim][CH3SO3]
to a more hydrophobic IL, [C4mim][CF3SO3], enhances the partition coefficients. Finally, the
sulfate anion is more effective in extracting vanillin than the sulfonate and acetate anions. Indeed,
methanesulfonate and acetate anions are strongly salting-out inducing ions, resulting therefore in
lower partition coefficients, and as observed before.[32]
In general it is observed that vanillin
extraction becomes more efficient using IL anions with a salting-in inducing behavior. Ions that
usually promote the solutes salting-in increase their partition coefficients in contrast to the salting-
out inducing ions that tend to decrease them. Salting-out inducing ions (high charge density ions)
have a greater tendency to form hydration complexes, increasing the surface tension of the cavity,
and thus decreasing the vanillin partition coefficient. On the other hand, for salting-in inducing ions
(low charge density ions) the tendency to form hydration complexes is marginal and thus they tend
to stabilize the solutes in solution by specific ion binding to the solute.[62, 65]
2.3.2. Effect of Temperature in Vanillin Partitioning
The effect of temperature on the partition coefficients of vanillin was studied with four ILs:
[C4mim]Cl, [C4mim][CH3SO4], [C7H7mim]Cl and [amim]Cl. These ILs allowed to study whether
the anion and cation suffer different influences under temperature variations. The temperatures
evaluated were 288.15 K, 298.15 K, 308.15 K, 318.15 K and 328.15 K. Accordingly to previous
studies,[11, 57, 112]
using IL-based ATPS, the temperature was shown to be either a negligible or a
significant factor in the extraction of various types of biomolecules. While He et al.[12]
indicated
that temperature had not significant influence on the distribution behavior of steroids, Pei et al.,[67]
on the other hand, reported that the temperature greatly influences the extraction efficiency of
proteins. As a result it can be established that the partition coefficients dependence on temperature
will largely depends on the solute and ternary system under study. The results obtained for the
temperature influence on the vanillin partitioning are shown in Figure 12.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
25
Figure 12: Partition coefficients of vanillin in IL + K3PO4 ATPS as a function of temperature for
the ILs: [C4mim]Cl, [C4mim][CH3SO4], [C7H7mim]Cl and [amim]Cl.
The results indicate that the temperature greatly influences the vanillin partition. This effect is less
pronounced for systems containing the IL [C4mim][CH3SO4]. The [C4mim][CH3SO4],
[C7H7mim]Cl and [amim]Cl systems have an optimum temperature for the extraction of vanillin at
298.15 K while for [C4mim]Cl the largest partition coefficient was observed at 308.15 K. Both IL
cation and anion contribute for the differences observed in the partition coefficients and their
dependence on temperature. The presence of maximum values in the partition coefficients as a
function of temperature suggest that the partitioning of vanillin is driven by opposite effects that
result from the temperature dependency of the energetic and entropic contributions.
For the systems studied the partition coefficients of vanillin are higher than those observed for the
system octanol-water[72]
that also show a decrease in the partition coefficients of vanillin with
temperature in the range between (298.15 and 318.15) K.
In order to calculate the vanillin thermodynamic parameters of transfer, such as the standard molar
Gibbs energy ( ), the standard molar enthalpy ( ), and the standard molar entropy of
transfer ( ) the van’t Hoff approach was used. The plots of ln(KVan) versus T-1 for the four ILs
studied, in the temperature range from 298.15 K to 328.15 K, are provided in Appendix C.
These parameters reveal the association equilibrium between the vanillin composition in two
different fluids. The following isochors were used to determine the molar thermodynamic functions
of transfer (Eqs. 2 to 4),[113-114]
[C4mim]Cl [C4mim][CH3SO4] [C7H7mim]Cl [amim]Cl
21.46
44.98 45.97
30.90
19.26
15.27
22.74 22.03
17.3514.63
29.25
44.19
30.52
27.09 26.99
33.74
36.45
21.83 22.03
13.55
0
10
20
30
40
50
288.15 298.15 308.15 318.15 328.15
KVan
T / K
0
mtrG 0
mtrH
0
mtrS
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
26
(2)
(3)
(4)
where KVan is the partition coefficient of vanillin, R is the universal gas constant (8.314 J·mol-1
·K-
1), T is the temperature (K), and , and are the standard molar enthalpy of
transfer, the standard molar entropy of transfer and the standard molar Gibbs energy of vanillin
transfer, respectively.
For the four systems, the plots of ln(Kvan) versus T-1 exhibit linearity indicating that the molar
enthalpy of transfer of vanillin is temperature independent. In Table 2, the obtained values of
, and at 298.15 K are summarized.
Table 2: Standard molar thermodynamic functions of transfer of vanillin at 298.15 K.
System ln(KVan)
[C4mim]Cl + K3PO4 + water -23.66 -46.51 -9.79 3.95
[C4mim][CH3SO4] + K3PO4 + water -12.62 -15.93 -7.87 3.18
[C7H7mim]Cl + K3PO4 + water -13.19 -13.47 -9.17 3.70
[amim]Cl + K3PO4 + water -24.09 -51.30 -8.80 3.55
The calculated
values are negative for all the systems evaluated reflecting thus the
spontaneous and preferential partitioning of vanillin for the IL-rich phase and as indicated by the
KVan > 1. values are negative indicating that the transference of vanillin from the K3PO4-
rich phase to the IL-rich phase is an exothermic process which further reflects the favorable
vanillin-IL type interactions. The standard molar enthalpies of transfer largely depend on the IL
anion while the effect of changing the IL cation is only relevant for cases where the chain side
cation is highly complex, as for [C7H7mim]Cl. These results again suggest that the partitioning
process is essentially controlled by the anion interactions with the solute.
The results here obtained show that the effect of temperature on the extraction of vanillin is highly
significant and that it is necessary to control the temperature at which the extraction is performed to
achieve the maximum efficiency.
R
S
TR
HK
0
mtr
0
mtrVan
1)ln(
0
mtr
0
mtr
0
mtr STHG
)ln( Van
0
mtr KRTG
0
mtrH 0
mtrS 0
mtrG
0
mtrG 0
mtrH 0
mtr S
1-
0
mtr
molkJ
H11-
0
mtr
molJ
K
S1-
0
mtr
molkJ
G
0
mtrG
0
mtrH
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
27
2.3.3. Effect of Concentration in Vanillin Partitioning
Figure 13 shows the partition coefficients for different initial concentrations of vanillin. The ILs
used to study this effect were [C4mim]Cl, [C4mim][CH3SO4] and [C7H7mim]Cl. The range of
vanillin concentrations in the water mass fraction at the ternary system varied between 0.5 g·dm-3
(3.3 × 10-3
mol·dm-3
) and 7.5 g.dm-3
(4.9 × 10-2
mol·dm-3
). It should be pointed out that the
saturation of vanillin in water at 298.15 K is around 10.0 g·dm-3
(6.5 × 10-2
mol·kg-1
).[1]
Figure 13: Partition coefficients of vanillin in IL + K3PO4 ATPS as a function of initial vanillin
concentration for the ILs: [C4mim]Cl, [C4mim][CH3SO4] and [C7H7mim]Cl.
For the three ILs studied there is an increase in the vanillin partition coefficients with the initial
concentration of the solute. Note that the value at 7.5 g·dm-3
is not shown for [C4mim][CH3SO4]
since the precipitation of vanillin/ionic liquid mixture was observed at these conditions (as
confirmed by NMR spectroscopic analysis). The precipitation of vanillin was found to be
of 82 wt % and results from the salting-out inducing ability of [C4mim][CH3SO4] as
discussed before. The dependence on the initial concentration is less pronounced for the IL
[C4mim][CH3SO4], while highly noticeable with [C4mim]Cl and [C7H7mim]Cl. These results
suggest that the IL cation starts to influences the dependency of the partitioning of vanillin with the
solute content. Since anions are typically more polarizable than cations, due to their more diffuse
valence electronic configuration, their hydration is usually stronger than that of cations and, as a
result, their salting-out effects are more prominent.[62, 65]
Thus, due to the stronger ability of anions
for salting-out, and particularly of the methylsulfate anion, the presence of additional vanillin does
not conduct to favourable interactions between IL anions and the solute. In contrast, methylsulfate
preferentially forms hydration complexes. Therefore, when increasing the vanillin content, the
[C4mim]Cl [C4mim][CH3SO4] [C7H7mim]Cl
33.09
44.98
57.02
88.3098.08
17.97 22.74
35.04 36.19
21.31
44.19
60.00
79.58
98.69
0
20
40
60
80
100
120
0.5 1.0 2.5 5.0 7.5
KVan
[Van] /(g·dm-3)
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
28
increase in the partition coefficients is more evident when changing the IL cation due to their
higher aptitude for specific binding with the phenolic aldehyde (typical salting-in inducing ions).
2.3.4. Density and Viscosity
The physical properties of the upper and lower phase in different ternary systems at various
compositions and temperatures are required for the design and scale up of extraction processes.
Therefore, densities and viscosities of all systems evaluated in this work, in the temperature range
between 298.15 K and 328.15 K, were determined at the following ternary composition: 15 wt %
of K3PO4 + 25 wt % of IL + 60 wt % of water (except for [OHC2mim]Cl that was at 15 wt % of
K3PO4 + 40 wt % of IL + 45 wt % of water). Results are displayed in Figures 14 to 17 and the
experimental data obtained are reported in Appendix B. In Figure 14, data for viscosities of the
chloride-based ILs are presented.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
29
Figure 14: Experimental viscosity (η) for the IL-rich phase (full symbols) and K3PO4-rich phase
(open symbols) for systems composed by chloride-based ILs + K3PO4 + H2O as a function of
temperature.
For the IL-rich phase the viscosities monotonically increase with the alkyl side chain length
increase from [C2mim]Cl (η = 2.98 mPa·s at 298.15 K) to [C10mim]Cl (η = 14.19 mPa·s at 298.15
K). Concerning the influence of the functional groups inclusion, the viscosities of the IL-rich phase
1
3
5
7
9
295 300 305 310 315 320 325 330
η/ m
Pa
.s
T / K
1.4
1.8
2.2
2.6
3.0
3.4
3.8
290 300 310 320 330
[C7H7mim]Cl [OHC2mim]Cl [amim]Cl [C4mim]Cl
1
3
5
7
9
11
13
15
295 300 305 310 315 320 325 330
η/
mP
a.s
T / K
[C2mim]Cl [C4mim]Cl [C6mim]Cl [C7mim]Cl [C10mim]Cl
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
30
decrease in the order: [OHC2mim]Cl (η = 6.27 mPa.s at 298.15 K) > [C7H7mim]Cl > [C4mim]Cl >
[amim]Cl (η = 2.96 mPa·s at 298.15 K). Although [OHC2mim]Cl presents relatively high viscosity
values it should be remarked that this system is richer in IL than the remaining systems so that a
direct comparison is not indeed fair. Regarding the inorganic salt-rich phase for the systems with
the chloride-based ILs the values of viscosities at 298.15 K range between 9.25 mPa.s for
[OHC2mim]Cl and 2.56 mPa.s for [C10mim]Cl. An opposite trend with the ILs was observed for
the viscosities at the K3PO4-rich phase. The opposite trends are in good agreement with each other
since the most viscous ILs are also the least water soluble.
The viscosities of the IL-rich phases are surprisingly low for ATPS and also sometimes lower than
the viscosities observed at the K3PO4-rich phase. This trend was observed for systems composed by
[C2mim]Cl, [C4mim]Cl, [OHC2mim]Cl and [amim]Cl. In particular for [C4mim]Cl there is an
inversion on the relative viscosities with the temperature.
The viscosity data for [C4mim]-based ILs are depicted in Figure 15.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
31
Figure 15: Experimental viscosity (η) for the IL-rich phase (full symbols) and K3PO4-rich phase
(open symbols) for systems composed by [C4mim]-based ILs + K3PO4 + H2O as a function of
temperature.
The results for the IL-rich phase show that the [C4mim][CF3SO3] system (η = 8.05 mPa·s at 298.15
K) presents the higher viscosity, while [C4mim][Br] (η = 3.23 mPa·s at 298.15 K) presents the
lower viscosity values. The viscosity data at 298.15 K, and at the same mass fraction compositions,
decrease in the following order: [C4mim][CF3SO3] > [C4mim][CH3CO2] > [C4mim]Cl >
[C4mim][CH3SO3] > [C4mim][N(CN)2] > [C4mim][CH3SO4] > [C4mim]Br. The viscosities of the
[C4mim]Br [C4mim][CH3SO4] [C4mim][CH3SO3] [C4mim][N(CN)2]
1
2
3
4
295 300 305 310 315 320 325 330
η/
mP
a.s
T / K
[C4mim][CF3SO3] [C4mim]Cl [C4mim][CH3CO2]
1
3
5
7
9
295 300 305 310 315 320 325 330
η/
mP
a.s
T / K
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
32
salt-rich phase at 298.15 K range between 4.76 mPa·s for [C4mim][CH3CO2] and 2.00 mPa·s for
[C4mim][CF3SO3]. Again, and as observed with the chloride-based ILs, the viscosity of the
inorganic salt-rich phase is higher for the systems containing the ILs [C4mim][CH3SO3] and
[C4mim][CH3CO2].
One of the critical problems related to the polymer-based ATPS is the high viscosity of the
polymer-rich phases, for example, at 293.15 K, for an aqueous solution composed by 22 wt % of
PEG2000 and 10 wt % of K3PO4, the viscosity takes the value of 18.94 mPa.s, while for an
aqueous solution of 19 wt % of PEG4000 and 9 wt % of K3PO4 the viscosity reaches 40.06
mPa.s.[115]
It was previously shown[61]
that phosphonium-based ATPS displayed lower viscosities
than polymer-based ATPS. As shown here the imidazolium-based ATPS present even lower
viscosity values which is highly beneficial in industrial processes. The low viscosity systems favors
the mass transfer of the solute between the two phases, as well as in improving the phases
handling.[116]
The density data for all the studied systems are presented in Figures 16 and 17, in the temperature
range between 298.15 K and 328.15 K. In all equilibrated systems the density of the K3PO4-rich
phase is higher than the density of the IL-rich phase. Only for the system containing
[C4mim][CF3SO3] the densities of both phases are similar.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
33
Figure 16: Experimental density (ρ) for the IL-rich phase (full symbols) and K3PO4-rich phase
(open symbols) for systems composed by chloride-based ILs + K3PO4 + H2O as a function of
temperature.
1.0
1.2
1.4
295 300 305 310 315 320 325 330
ρ/
g.c
m-3
T/ K
[C2mim]Cl [C4mim]Cl [C6mim]Cl [C7mim]Cl [C10mim]Cl
[C7H7mim]Cl [OHC2mim]Cl [amim]Cl [C4mim]Cl
1.0
1.2
1.4
1.6
295 300 305 310 315 320 325 330
ρ /
g.c
m-3
T / K
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
34
Figure 17: Experimental density (ρ) for the IL-rich phase (full symbols) and K3PO4-rich phase
(open symbols) for systems composed by [C4mim]-based ILs + K3PO4 + H2O as a function of
temperature.
For the IL-rich phase the values of densities at 298.15 K range between 1.0366 g·cm-3
for
[C4mim][N(CN)2] and 1.2169 g·cm-3
for [C4mim][CF3SO3]. For the bottom phase (K3PO4-rich
phase) the density values at 298.15 K range between 1.2291 g·cm-3
in the system with
[C4mim][CF3SO3] and 1.5265 g·cm-3
for the system composed by [OHC2mim]Cl. Comparing IL-
[C4mim][CF3SO3][C4mim]Cl [C4mim][CH3CO2] [C4mim][CH3SO3]
1.0
1.2
1.4
295 300 305 310 315 320 325 330
ρ/
g.c
m-3
T / K
[C4mim]Br [C4mim][CH3SO4] [C4mim][N(CN)2]
1.0
1.2
1.4
295 300 305 310 315 320 325 330
ρ /
g.c
m-3
T / K
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
35
based ATPS and typical polymer-inorganic salt ATPS[115]
there are not significant differences in
the density values. In both systems the top phase is the IL- or polymer-rich phase, while the bottom
layer is the inorganic salt-rich phase.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
36
2.4. Conclusions
In this work the optimization of the IL structure for the improved extraction of vanillin was
experimentally determined by measuring the partition coefficients on several IL-based ATPS. The
effect of the IL cation and anion nature, the temperature of equilibrium and the concentration of the
solute were evaluated. All the studied parameters have shown to influence the extraction of
vanillin.
For all the studied systems, and at all the conditions analyzed, vanillin preferentially partitions for
the IL-rich phase presenting KVan > 1. The partition coefficients dependency with the cation alkyl
chain length displays a maximum for the system formed by [C6mim]Cl resulting from a decrease in
the polar character of the IL cation and lower surface tension at the IL-rich phase. The introduction
of features such as double bounds, benzyl and hydroxyl groups in ILs had only a marginal impact
on the partition coefficients of vanillin. Regarding the IL anion, vanillin partitions preferentially for
ILs composed by halogenated anions, such as Cl- or Br
-, or by anions with a higher hydrogen
bonding accepting character, such as [N(CN)2]-.
The influence of temperature in the partitioning of vanillin presented a maximum in the extraction
efficiency at 298.15 K for [C4mim][CH3SO4], [C7H7mim]Cl and [amim]Cl and at 308.15 K for
[C4mim]Cl. Moreover, the partition coefficients of vanillin increased monotonically with the initial
concentration of the solute added to the global system. Variations in the partition coefficients as a
function of the vanillin concentration were more dependent on the IL cation nature.
The viscosities of the IL-rich phase in all IL-based ATPS studied were found to be substantially
lower than those observed in typical polymer-based ATPS what constitutes an additional advantage
for industrial applications of the systems here studied.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
37
3. Extraction of gallic
acid in ATPS with ILs
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
38
3.1. Gallic Acid
Gallic acid, 3,4,5-trihydroxybenzoic acid, with the chemical formula C6H2(OH)3COOH, is a polar
compound and its physical appearance is as yellowish-white crystals. Table 3 depicts some
thermophysical properties of this phenolic compound.
Table 3: Thermophysical properties of gallic acid.[92, 117]
Molar mass
(g·mol-1
)
Density
(g·cm-3
)
Melting point
(K)
Solubility in water
(g·dm-3
)
pKa
170.12 1.7 at 298.15 K 523 11 at 293.15 K 4.41 at 298 K
Gallic acid, whose molecular structure is reported in Figure 18, is present in diverse natural
products, such as fruits (grapes[118]
), pomegranate husk,[119]
vegetables,[75]
green and black teas,[78]
oak wood,[120]
and some plants like Plantae regnum.[121]
Besides its natural sources, gallic acid can
also be found in residual waste. In olive mill effluents, gallic acid, protocatechuic acid and vanillic
acid are the second major family of PhCs available[1]
. Gallic
acid can be either found in its free form or in the tannins
constitution. Nevertheless, in wood, gallic acid is present in
the form of gallotannins (hydrolysable tannins) and the
further contact of such hydrolysable tannins with acidic
media or hydrolytic enzymes (e.g. tannase) originate sugars
and phenolic acids,
such as gallic acid.[78, 122-123]
The
extraction of this biomolecule has a great practical interest
because it has several important biological characteristics. It
has properties of antioxidant, anti-inflammatory, antifungal,
anti-tumor, diuretic, depurative, intestinal antiseptic, bacteriostatic and bactericidal, tonic, anti-
arthritic and as an hydrogen carrier.[5, 118, 121, 124-125]
Gallic acid also displays an important role as a
metabolic inhibitor of some microorganisms (antimicrobial action). Due to these attractive
characteristics associated to gallic acid, this biomolecule plays an essential position in the
pharmaceutical industry in the treatment of gastric tonus problems, anorexia, bloating, gases,
urinary diseases gout, haemostatic, skin repairer and sedative since it is well absorbed by
humans.[75, 122] Moreover, gallic acid is commonly used as antioxidant in food additives.
[4] It has a
percentage antioxidant activity of 92.92 %.[74]
The utilization of natural phenolic compounds for
nutraceutical and cosmetic applications has proved to be highly advantageous compared to
synthetic substitutes that could demonstrate adverse effects.[125]
Figure 18: Chemical structure of
gallic acid.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
39
Being an antioxidant, gallic acid in the presence of alkaline solutions gets a brownish color due to
absorption of oxygen,[121]
and can be further degraded during the extraction procedures. To prevent
the degradation of gallic acid another antioxidant can be added to the system of extraction, such as
ascorbic acid.
Gallic acid was found to be partially soluble in different solvents and the order of solubility in
several solvents is as follows: methanol > ethanol > water > ethyl acetate.[122]
This solubility in
several common and molecular solvents represents an important advantage from the industrial
point of view.[121-122] Furthermore, studies regarding the effect of salts on the solubility of phenolic
compounds,[1]
showed that there is a decrease in the solubility of gallic acid with increasing the salt
molality, where the influence of the cation was shown to be highly noticeable.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
40
3.2. Experimental section
Before the studies of extraction of gallic acid by aqueous two-phase systems composed by Na2SO4
and different ILs, it was necessary to determine the respective ternary phase diagrams due to their
absence in literature. These diagrams are useful to know in which conditions was possible to form a
biphasic mixture and at which ternary compositions the extraction can be performed.
3.2.1. Chemicals
The ILs studied to determine the ternary phase diagrams were: 1-butyl-3-methylimidazolium
chloride, [C4mim]Cl; 1-hexyl-3-methylimidazolium chloride, [C6mim]Cl; 1-heptyl-3-
methylimidazolium chloride, [C7mim]Cl; 1-methyl-3-octylimidazolium chloride, [C8mim]Cl; 1-
butyl-3-methylimidazolium bromide, [C4mim]Br; 1-butyl-3-methylimidazolium methanesulfonate,
[C4mim][CH3SO3]; 1-butyl-3-methylimidazolium acetate, [C4mim][CH3CO2]; 1-butyl-3-
methylimidazolium methylsulfate [C4mim][CH3SO4]; 1-butyl-3-methyl-imidazolium
trifluoromethanesulfonate, [C4mim][CF3SO3]; 1-butyl-3-methylimidazolium dicyanamide,
[C4mim][N(CN)2]; 1-butyl-3-methylimidazolium hydrogenosulfate, [C4mim][HSO4]; 1-butyl-3-
methylimidazolium tosylate, [C4mim][TOS]; 1-ethyl-3-methylimidazolium methylsulfate
[C2mim][CH3SO4]; 1-ethyl-3-methyl-imidazolium trifluoromethanesulfonate, [C2mim][CF3SO3]; 1-
benzyl-3-methylimidazolium chloride, [C7H7mim]Cl; 1-benzyl-3-methylimidazolium ethylsulfate,
[C7H7mim][C2H5SO4]; 1-allyl-3-methylimidazolium ethylsulfate, [amim][C2H5SO4]; 1-butyl-3-
methylimidazolium ethylsulfate, [C4mim][C2H5SO4]; 1-butyl-3-methylpyridinium chloride,
[C4mpy]Cl(1,3); 1-butyl-3-methylpiperidinium chloride, [C4mpip]Cl; 1-butyl-3-
methylpyrrolidinium chloride, [C4mpyrr]Cl; 1-octylpyridinium dicyanamide, [C8py][N(CN)2]. All
ILs were supplied by Iolitec. To reduce the impurities content to negligible values, ILs individual
samples were purified under constant agitation at vacuum and moderate temperature (353 K) for a
minimum of 24 hours. After this step, the purity of each IL was checked by 1H,
13C and
19F NMR
spectra and found to be > 99 wt % for all samples. The inorganic salt Na2SO4 was from LabSolve
(purity > 99.8 wt %), and gallic acid, 99.5 wt % pure, was from Merck. The molecular structure of
gallic acid is depicted in Figure 17. The water employed was double distilled, passed across a
reverse osmosis system and further treated with a Milli-Q plus 185 water purification apparatus.
The structures of ILs that were successful in the determination of the phase diagrams are shown in
Figure 19.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
41
3.2.2.
N+
N
S OO
O-
F
F
F
N+
N
Cl-
N+
NCl-
N+
N
Br-
S
O
O
-O
O
N+
N
N
N N
N+
N
S
O
OO
O
N+
N
N+
N
S
O
OO
O
N+
N
N+
N
N N
N+
N
S OO
O-
F
F
F
i
ii
iii
iv
v vi
viiviii
ix
x
xi
xii
N+
NO
S
O
O
Cl-
Figure 19: Chemical structure of the studied ILs: (i) [C7mim]Cl; (ii) [C8mim]Cl; (iii)
[C4mim]Br; (iv) [C4mim][CH3SO4]; (v) [C4mim][CF3SO3]; (vi) [C4mim][N(CN)2]; (vii)
[C2mim][CF3SO3]; (viii) [C7H7mim] [C2H5SO4]; (ix) [C4mim][TOS]; (x) [C4mim] [C2H5SO4];
(xi) [C8py][N(CN)2]; (xii) [C7H7mim]Cl.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
42
3.2.2. Experimental procedure
3.2.2.1. Preparation of Phase Diagrams
Aqueous solutions of Na2SO4 and aqueous solutions of the different hydrophilic ILs were prepared
gravimetrically as described in Appendix D. The phase
diagrams were determined at 298 K (± 1 K) and at
atmospheric pressure through the cloud point titration
method.[61]
Drop-wise addition of the aqueous inorganic
salt solution to each IL aqueous solution was carried out
until the detection of a cloudy (biphasic solution),
followed by drop-wise addition of ultra-pure water until
the finding of a clear and limpid solution (monophasic
region), as shown in Figure 20. Drop-wise additions were
carried out under constant stirring. The ternary systems
compositions were determined by the weight
quantification of all components within an uncertainty of
± 10-5
g.
For [C8mim]Cl it was not possible to determine the complete binodal curve using the described
method, so the turbidometric titration method was used in parallel.[126]
Test tubes at the biphasic
region with different compositions of known weight of IL, salt and water were prepared. Then, it
was drop-wise added pure water, under continuous stirring, until reaching a limpid solution (single-
phase region). The total amount of added water was determined gravimetrically and the mass
fraction of each component in the mixture was recalculated.
3.2.2.2. Determination of Tie-Lines
Tie-lines (TLs) were determined by a gravimetric method originally proposed by Merchuck et
al.[127]
For the determination of TLs, a mixture at the biphasic region was gravimetrically prepared
with Na2SO4 + water + IL, vigorously agitated, and left to equilibrate for at least 12 h at 298 K,
aiming at completing separating both phases. After a careful separation step, both top and bottom
phases were weighed. Finally, each TL was determined by mass balance through the application of
the lever rule to the relationship between the top mass phase composition and the overall system
composition. The experimental binodal curves were fitted using Equation 5:[127]
)]()exp[( 35.0 CXBXAY (5)
Figure 20: Experimental
determination of the binodal curves
for the aqueous systems IL-Na2SO4:
in the first picture it is shown a limpid
and clear solution while in the second
picture it denotes a cloudy solution.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
43
where Y and X are the IL and salt weight percentages, respectively, and A, B and C are constants
obtained by the regression.
For the TL determination the following system of four equations (Equations 6 to 9) and four
unknown values (YT, YB, XT and XB) was solved:[127]
)]()exp[( 35.0
TTT CXBXAY (6)
)]()exp[( 35.0
BBB CXBXAY (7)
BM
T YY
Y
1 (8)
BM
T XX
X
1 (9)
where T, B and M designate the top phase, the bottom phase and the mixture, respectively. X and Y
represent, respectively, the weight fraction of inorganic salt and IL, and α is the ratio between the
mass of the top phase and the total mass of the mixture. The system solution results in the
concentration (wt %) of the IL and inorganic salt in the top and bottom phases, and thus, TLs can
be simply represented.
For the calculation of the tie-lines length (TLL) it was employed Equation 10:
22 )()(TLL BTBT YYXX (10)
where T and B symbolize, respectively, the top and bottom phases, and X and Y are the weight
fraction of inorganic salt and IL, respectively.
3.2.2.3. Partitioning of Biomolecules
The ternary mixtures compositions were chosen based on the determined phase diagrams
containing each of the ILs. A ternary mixture was prepared within the biphasic region containing
15 wt % of inorganic salt, 60 wt % of an aqueous solution of gallic acid with 0.5 g·dm-3
(3.06×10-3
)
and 25 wt % of selected ILs. The ternary compositions were prepared by weight with an uncertainty
of ± 10-5
g. Each ternary mixture (IL, inorganic salt and aqueous solution of gallic acid) was
prepared in small ampoules (10 cm3) especially built for such extraction procedures and vigorously
stirred. After such step, the ampoules were kept in rest for at least 12 h, and at 298.15 ± 0.01 K in
an air bath equipped with a Pt 100 probe and a temperature controller, to reach the equilibrium and
the separation of both phases. A preliminary study showed that the equilibration of gallic acid was
completely attained after a period of 12 h. The time required to establish the equilibrium of gallic
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
44
acid was experimentally determined by measuring the concentration of gallic acid in each phase at
different times until reproducible data were obtained.
After a gentle separation of both phases, the amount of gallic acid at each aqueous phase was
quantified through UV-spectroscopy, using a SHIMADZU UV-1700, Pharma-Spec Spectrometer,
at wavelength of 262 nm. Calibration curves were properly established and are reported in
Appendix A. At least three individual samples of each phase were quantified in order to determine
the gallic acid partition coefficients and the respective standard deviations. Possible interferences
of both inorganic salts and all ILs with the analytical method were investigated and found to be
slightly significant at the wavelength of 262 nm and at the magnitude of the dilutions carried.
Mixtures at the same fraction composition were prepared, for each of the ILs, using pure water
instead of an aqueous solution of gallic acid, and the proper discount in the analytical quantification
was performed.
The partition coefficients of gallic acid, KGA, were determined as the ratio of the concentration of
gallic acid in the IL and in the inorganic salt (Na2SO4, K3PO4 or K2HPO4/KH2PO4) aqueous-rich
phases, accordingly to:
X
ILGA
GA
GAK (11)
where [GA]IL and [GA]x are the concentration of gallic acid in the IL and in each of the inorganic
salt aqueous-rich phases, respectively. At the compositions used, the top layer is the IL-rich phase
while the bottom phase is the inorganic salt-rich phase. The only exceptions are the specific case of
the [C2mim][CF3SO3]-K3PO4, [C4mim][CF3SO3]-K3PO4 and [C4mim][CF3SO3]-Na2SO4 systems
where it was observed an inversion on the density of both phases.
3.2.2.4. pH measurement
The pH of the both IL and inorganic salt-rich phases was measured at 298 K using an HI 9321
Microprocessor pH meter (HANNA instruments) within an uncertainty of ± 0.01.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
45
3.3. Results and Discussion
3.3.1. Phase Diagrams
Since the partitioning of gallic acid could be somewhat affected by the pH of the solutions, several
inorganic salts in combination with distinct IL-based ATPS were investigated. The inorganic salt
most used was Na2SO4 because it is normally used in the acidic hydrolysis for breaking the
hemicellulose fraction of lignocellulose into monosaccharides of vegetal biomass (wood), aiming at
a posteriori extract value-added compounds such as phenolic compounds, furfural and others.
Partial depolymerization of lignin and lignin-hemicellulose linkages occur through acid hydrolysis
and the acid-soluble lignin fraction is mainly identified with the presence of PhCs. In order to know
the degree of extraction with ILs in acidic systems, the extraction of gallic acid with Na2SO4-based
ATPS was studied more extensively.[128-129]
Due to the absence in literature of the ternary phase diagrams for IL-Na2SO4-water systems, they
were here determined. Table 4 represents all the evaluated systems. It should be pointed out that
with Na2SO4 it was not possible to determine the phase diagrams of all the proposed ILs due to the
lack of an immiscibility region. All systems signed with means that it was not possible to observe
a two phase region in the conditions studied. The systems with indicate the phase diagrams
studied. In some cases it was found a solid-liquid region instead of a liquid-liquid region.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
46
Table 4: Initial weight fraction compositions for the determination of the phase diagrams and
indication of the possibility of existing liquid-liquid equilibrium.
IL + Na2SO4 + Water system Weight fraction composition / wt %
LLE IL Na2SO4
[C2mim][CH3SO4] 59.94 27.00
[C2mim][CF3SO3] 59.17 24.89
[C4mim]Cl
79.61 29.98
78.38 29.98
69.70 29.98
79.43 24.17
70.05 24.17
63.73 24.08
54.80 24.08 49.69 24.08
[C4mim]Br 59.40 24.89
[C4mim][CH3CO2] 62.06 24.89
[C4mim][CF3SO3] 59.41 29.98
[C4mim][N(CN)2] 60.45 30.26
[C4mim][HSO4] 60.05 26.64
[C4mim][CH3SO4] 59.46 30.26
[C4mim][C2H5SO4] 60.83 27.00
[C4mim][TOS] 59.43 30.26
[C6mim]Cl 61.52 24.89
[C7mim]Cl 59.27 26.12
[C8mim]Cl 60.15 24.89
[amim][C2H5SO4] 63.51 24.89
[C7H7mim]Cl 78.64 29.98
60.68 24.50
[C7H7mim][C2H5SO4] 60.36 24.50
[C4mim][DMphosp] 58.74 26.64
[C4mpyrr]Cl 59.85 30.26
46.60 30.26
[C4mpip]Cl 59.19 26.64
[C4mpy]Cl 59.58 24.89
[C8py][N(CN)2] 60.42 26.12
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
47
From an inspection to Table 4, it is visible that anions or cations with more hydrophobic
characteristics have a greater capacity to promote ATPS. Examples are those systems with IL
containing the anions [N(CN)2]- and [CF3SO3]
- or larger alkyl side chains at the cation. Figure 21
depicts the experimental phase diagrams, at 298 K and at atmospheric pressure, for the biphasic
systems determined with Na2SO4 + water + different ILs. The phase diagrams are presented in
molality units for a more comprehensive perception of the impact of distinct ILs through ATPS
formation.
Figure 21: Ternary phase diagrams for all the ILs studied at 298 K and atmospheric pressure.
The presence of inorganic salt/IL combinations turns ATPS more complex than typical PEG-based
ATPS. Thus a more complex equilibrium is expected taking into account that ion exchange and
ion-pairing phenomena may occur.[32, 49]
Although, it was previously found that electroneutrality is
[C4mim][C2H5SO4][C4mim][TOS] [C4mim][CH3SO4] [C4mim][N(CN)2]
[C4mim][CF3SO3] [C7mim]Cl [C8py][N(CN)2]
X [C2mim][CF3SO3]
[C7H7mim][C2H5SO4]
[C4mim]Br
+ [C7H7mim]Cl[C8mim]Cl
0
2
4
6
8
0.0 0.5 1.0 1.5 2.0
[IL
] /
(mol·
kg
-1)
[Na2SO4] / (mol·kg-1)
Biphasic Region
Monophasic
Region
0
2
4
0.1 0.3 0.5 0.7 0.9
[IL
] /
(mo
l·k
g-1
)
[Na2SO4] / (mol·kg-1)
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
48
maintained in such systems and that the ion partitioning is in the order of the deviation aroused
from the cloud point titration method.[69]
Binodal curves with a larger biphasic region are an indication of the higher capability by ILs to
promote ATPS, that is, ILs with stronger salting-in inducing behavior. From Figure 21 the
tendency of ILs to promote ATPS with Na2SO4 follows the order: [C8py][N(CN)2] >
[C4mim][CF3SO3] > [C4mim][TOS] > [C4mim][N(CN)2] > [C2mim][CF3SO3] ≈
[C7H7mim][C2H5SO4] > [C4mim][C2H5SO4] > [C4mim][CH3SO4] ≈ [C4mim]Br > [C7H7mim]Cl >
[C7mim]Cl ≈ [C8mim]Cl. Analyzing the anion influence, when fixing the cation [C4mim]+, the
ability of the IL anions to promote ATPS follows the rank: [CF3SO3]- > [TOS]
- > [N(CN)2]
- >
[C2H5SO4]- > [CH3SO4]
- > Br
-. Similarly, with the common cation [C7H7mim]
+, [C2H5SO4]
- shows a
higher ability to induce ATPS than Cl-. In spite of a different inorganic salt, this trend on the IL
anions agrees with the one reported previously.[32]
This rank reflects the competition between the
inorganic salt and the IL ions for the formation of water-ion hydration complexes. Thus, the ability
of the anions’ hydrogen bonding accepting strength is determinant for the ATPS formation. Indeed,
in a previous work it was shown that the IL inducing capacity to promote ATPS follows the anion
hydrogen bond basicity[32]
, and additionally corroborated by this work, employing a different
inorganic salt.
Moreover, an increase in the alkyl chain length in both the IL cation or anion enhances the ability
of the IL for ATPS formation, as deduced when comparing the two following pairs of ILs:
[C2mim][CF3SO3] and [C4mim][CF3SO3], and [C4mim][CH3SO4]
and [C4mim][C2H5SO4].
Obviously, this trend can be justified due to an increase in the IL hydrophobicity generated by an
increase in the alkyl chain lengths. Nevertheless, for systems with longer alkyl chains, [C7mim]Cl
and [C8mim]Cl, the tendency of ILs for ATPS formation is very close, although [C8mim]Cl is
slightly more favorable to create ATPS. This small difference with longer alkyl chain lengths can
be related with the formation of IL aggregates in aqueous solutions increasing thus the ILs affinity
for water.[110]
Comparing both [C4mim][C2H5SO4] and [C7H7mim][C2H5SO4], it is verified that the
former presents a higher ability to promote ATPS, and which is in agreement with literature when
using a different anion.[49]
The manipulation of the phase diagrams through the IL cation/anion combinations clearly reflects
the ILs character of "designer solvents". Therefore, an appropriate selection of cation or anion
could provide ATPS with specific phase behavior.
All experimental data were correlated by Equation 5. The adjusted parameters for all ternary
systems studied are presented in Table 5.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
49
Table 5: Adjusted Parameters used to describe the experimental binodal data by Equation 5.
IL + Na2SO4 +
Water system A B 10
5C R
2
[C2mim][CF3SO3] 106.1 ± 2.1 -0.439 ± 0.010 16.2 ± 2.0 0.9990
[C4mim][CF3SO3] 130.0 ± 15.7 -0.760 ± 0.066 30.0 ± 10.0 0.9616
[C4mim]Br 97.5 ± 2.6 -0.393 ± 0.016 1.0 ± 5.8 0.9989
[C4mim][N(CN)2] 88.8 ± 1.0 -0.446 ±0.006 20.2 ± 0.7 0.9963
[C4mim][CH3SO4] 105.4 ± 2.4 -0.396 ± 0.009 4.4 ± 0.4 0.9980
[C4mim][C2H5SO4] 95.4 ± 0.7 -0.393 ± 0.004 5.7 ± 0.2 0.9997
[C4mim][TOS] 101.5 ± 0.7 -0.417 ± 0.003 18.7 ± 0.2 0.9995
[C7mim]Cl 91.6 ± 2.0 -0.287 ± 0.011 9.9 ± 1.5 1.0000
[C7H7mim]Cl 104.7 ± 7.4 -0.372 ± 0.036 4.9 ± 6.6 0.9982
[C7H7mim][C2H5SO4] 100.6 ± 1.6 -0.383 ± 0.007 12.9 ± 0.5 0.9997
[C8mim]Cl 84.6 ± 6.2 -0.245 ± 0.033 13.7 ± 3.1 0.9984
[C8py][N(CN)2] 195.2 ± 2.4 -1.079 ± 0.008 9.9 ± 2.3 0.9984
The correlation of the experimental data through the application of Equation 5, as well as the
graphical representation of the TLs measured, are presented in Figure 22. Experimental data with
the phases composition at equilibrium, and respective TLLs, are reported in Appendix E.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
50
0
10
20
30
40
50
60
70
0 10 20 30 40
% w
t [C
4m
im][
N(C
N) 2
]
% wt Na2SO4
0
10
20
30
40
50
60
0 10 20 30
% w
t [C
4m
im][
CH
3S
O4]
% wt Na2SO4
0
10
20
30
40
50
60
0 10 20 30 40
% w
t [C
4m
im][
C2H
5S
O4]
% wt Na2SO4
0
10
20
30
40
50
60
70
80
0 10 20 30 40
% w
t [C
4m
im][
TO
S]
% wt Na2SO4
0
10
20
30
40
50
0 10 20 30
[C7m
im]C
l
% wt Na2SO4
0
10
20
30
40
50
60
70
80
0 10 20 30
[C2m
im][
CF
3S
O3]
% wt Na2SO4
0
10
20
30
40
50
60
70
80
0 10 20 30
[C8p
y][
N(C
N) 2
]
% wt Na2SO4
0
20
40
60
0 10 20 30
[C7H
7m
im][
C2H
5S
O4]
% wt Na2SO4
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
51
Figure 22: Phase diagrams for the different ternary systems composed by IL+ Na2SO4+ H2O at 298
K and atmospheric pressure: ♦, experimental binodal data; □ ,TL data; ▬ fitting of experimental
data by the method proposed by Merchuck et al.[127]
For all systems represented, except for [C4mim][CF3SO3], the top-rich phase is the IL-rich phase
while the bottom phase is the Na2SO4-rich phase. Observing all the binodal curves and respective
tie-lines it can be seen that the IL concentration in the bottom phase is very small, and in some
cases, the IL is almost completely excluded from that phase. Additionally, for systems with more
than one TL, the TLs slope are not strictly parallel. Although, these deviations in the TLs slopes are
in conformity with literature.[49]
This fact occurs particularly for longer TLLs.
3.3.2. Effect of IL ions and pH in the acid gallic partitioning
The extraction of a molecule in ATPS based on ILs depends on its ability to migrate for the IL-rich
phase. In this study it was assessed the selectivity of gallic acid for different ILs and how the pH of
the medium influences the respective partition coefficients. In order to evaluate the IL ions
influence in the extraction of gallic acid, several combinations between cations and anions were
performed. In addition, to evaluate the pH of the medium several inorganic salts combinations were
used. The mass fraction compositions used for the determination of each partition coefficient,
0
10
20
30
40
50
60
0 10 20 30 40 50
%w
[C
4m
im][
CF
3S
O3]
%w Na2SO4
0
10
20
30
40
50
60
0 10 20 30
% w
t [C
4m
im]B
r
% wt Na2SO4
0
10
20
30
40
50
60
0 10 20 30
[C7H
7m
im]C
l
% wt Na2SO4
0
10
20
30
40
50
0 10 20 30
wt %
[C
8m
im]C
l
wt % Na2SO4
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
52
partition coefficients values and respective standard deviations, and pH values are reported in
Appendix F.
For all the studied systems it was observed that specific interactions between gallic acid and
imidazolium-based ILs are favorable since gallic acid preferentially migrates for the IL-rich phase
(KGA > 1). However, in general the partition coefficients were lower than those obtained with
vanillin for the same systems (Chapter 2 of this thesis).
The partition coefficients of gallic acid in aqueous biphasic systems composed by Na2SO4 and
distinct ILs were measured at 298.15 K and are presented in Figure 23. From the inspection of
Figure 23 it can be seen that KGA ranges between 10.25 and 29.58 and thus depend on the IL used.
The partition coefficients decrease in the following order: [C4mim][C2H5SO4] >> [C7mim]Cl >
[C4mim][N(CN)2] > [C4mim][CH3SO4] ≈ [C4mim][CF3SO3] > [C8mim]Cl > [C4mim]Br >>
[C2mim][CF3SO3].
Figure 23: Partition coefficients of gallic acid for different ILs + Na2SO4 ATPS at 298.15 K.
Ranging from [C2mim][CF3SO3]- to [C4mim][CF3SO3]-based systems (increase of size of the alkyl
side chain of the imidazolium cation) leads to an increase in the partition coefficient values. This
fact results from the concomitant increase of the IL free volume while decreasing the surface
tension of the system.[49, 109]
In parallel, an increase in the alkyl chain at the anion, from
[C4mim][CH3SO4] to [C4mim][C2H5SO4], promotes the increase of the partition coefficients for
gallic acid. For the systems composed by [C7mim]Cl and [C8mim]Cl it was found a smaller
decrease on the partition coefficient values. Longer alkyl chains lead to a decrease in the coloumbic
and polar interactions and to an increase in dispersive-type interactions between the IL ions.[42, 111]
10.25
20.73
18.12
21.23 20.84
29.58
21.94
19.39
0
10
20
30
KGA
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
53
Fixing the [C4mim]+ cation, the IL anion effect on the gallic acid extraction follows the rank:
[C4mim][C2H5SO4] > [C4mim][N(CN)2] ≈ [C4mim][CH3SO4] ≈ [C4mim][CF3SO3] > [C4mim]Br.
Therefore, based on those data, anions with longer alkyl chains or higher hydrogen bond accepting
strength present higher extraction abilities for gallic acid. Nevertheless, the effect of the anion is
marginal and the higher influence was mainly observed with the increase of the alkyl chain length
in [C4mim][C2H5SO4].
The pH of the aqueous medium in the extraction of gallic acid was evaluated using different
inorganic salts. The gallic acid partition coefficients obtained are depicted in Figure 24. The pH of
each aqueous rich phase are presented in Table 6. In addition, the mass fraction compositions used
for the determination of each partition coefficient, partition coefficients values and respective
standard deviations, and pH values are presented in Appendix F.
Figure 24: Partition coefficients of gallic acid in IL + different inorganic salt ATPS for the ILs:
[C2mim][CF3SO3], [C4mim][CF3SO3] and [C7mim]Cl.
From Figure 24, the choice of the inorganic salt employed, and hence of the pH of the system, is
highly important and shows to be more significant than the IL used. Although K3PO4 is a stronger
salting-out agent than Na2SO4 it is here obvious that the influence of the pH of the aqueous medium
plays a major role since higher partition coefficients of gallic acid are attained with ATPS
employing Na2SO4. For instance, focusing on the partition coefficients of the extraction system
composed by [C4mim][CF3SO3] the partition coefficients range between 0.18 and 20.73 for salts
K3PO4 and Na2SO4, respectively.
For all the studied inorganic salts, the IL that presents an higher ability for extracting gallic acid is
[C7mim]Cl. Nevertheless, since the biphasic region for the system [C7mim]Cl + K2HPO4/KH2PO4 +
10.25
20.7321.94
0.91 0.76
11.59
0.58 0.18
8.16
0
10
20
KGA
[C2mim][CF3SO3] [C4mim][CF3SO3] [C7mim]Cl
Na2SO4 K2HPO4/ KH2PO4 K3PO4
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
54
water is smaller than for the outstanding ILs, the concentration of this ILs was slightly higher and a
direct comparison between [C7mim]Cl and the remaining ILs is not strictly accurate. While for the
ILs [C2mim][CF3SO3] and [C4mim][CF3SO3] their mass composition was around 25 wt %, for the
[C7mim]Cl + K2HPO4/KH2PO4 system the IL was at approximately 30 wt %. Therefore, aiming at
better understanding the effect of the IL concentration, the system [C4mim][CF3SO3] +
K2HPO4/KH2PO4 was also evaluated with the IL at 30 wt % (Figure 25).
Figure 25: Partition coefficients of gallic acid in IL + K2HPO4/KH2PO4 ATPS for [C4mim][CF3SO3]
at 25 wt % and 30 wt %.
Actually there is an increase of the gallic acid partition coefficient with the increase of 5 wt % of
IL. However, at 30 wt % of IL, it is observed that the [C7mim]Cl has indeed a significantly higher
partition coefficient. From the inspection of Table 6, and from the results depicted in Figure 24 and
Figure 25, the acidity of the medium largely influences the gallic acid partitioning and favors the
solute migration for the IL-rich phase.
Table 6: pH values as function the different systems performed.
IL+inorganic salt + water Na2SO4 K2HPO4/KH2PO4 K3PO4
[C2mim][CF3SO3] Salt-rich phase 3.32 7.28 13.09
IL-rich phase 2.71 7.57 13.15
[C4mim][CF3SO3] Salt-rich phase 3.04 7.10 12.85
IL-rich phase 3.12 7.37 13.10
[C7mim]Cl Salt-rich phase 4.16 7.22 12.85
IL-rich phase 4.15 7.45 12.99
The pKa of gallic acid is 4.41, which means that in the system with salt Na2SO4, gallic acid is in its
anionic form. As a result, the anionic form preferentially migrates for IL-rich phases.
0.76
4.25
0
1
2
3
4
5KGA
wt % IL25 30
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
55
From the inspection of Table 6, it can be seen that there are no major deviations in the aqueous
phases pH at the salt-rich phase and IL-rich phase for the same system or IL. Comparing the pH
values for systems composed by [C2mim][CF3SO3] or [C4mim][CF3SO3] there are not large
differences in the pH values. Thus, the cation alkyl chain length does not significantly contributes
for differences in the pH values at the two aqueous phases in equilibrium. Nevertheless, changing
the anion from [CF3SO3]- to Cl
- guides to slightly differences in the acidity of both aqueous phases.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
56
3.4. Conclusions
In this part of the work ternary phase diagrams employing diverse ILs, water and Na2SO4 were
determined due to their absence in literature. Generally, it was observed that anions or cations with
more hydrophobic characteristics, ILs with stronger salting-in inducing behavior, have a greater
capability to promote ATPS. Additionally, all phase diagrams were adjusted through the equation
proposed by Merchuck et al.[127]
and supplementary TLs and TLLs for each system were
determined.
The ability to extract gallic acid of several IL-based ATPS was studied. For aqueous systems
composed by Na2SO4 and IL, it was verified that the increase of the alkyl side chain length, in both
cation and anion, leads to an increase in the partition coefficient values. Moreover, ILs presenting
anions with higher hydrogen bond accepting strength present higher extraction abilities for gallic
acid.
Comparing the extractions performed with ATPS containing different inorganic salts (Na2SO4,
K2HPO4/KH2PO4, K3PO4) it can be concluded that the Na2SO4 provides enhanced recovery of
gallic acid for the IL-rich phase. Therefore, the choice of the inorganic salt employed in IL-based
ATPS, and hence of the system pH, is highly important and shows to be more significant than the
IL used.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
58
In the future it would be interesting to extend this type of study to other phenolic compounds and
test more conditions in order to optimize extraction routes.
In order to better evaluate the effect of pH on the phenolic compounds extraction it will be
important to extend the study for more systems based on different ILs beyond those demonstrated
here. Also it is important to determine the pH of both rich phases for some systems already
evaluated in this work.
It would also be of utmost importance to use the knowledge acquired in this work to proceed to
more practical and real experiments attempting direct extractions from biomass, such as the
extraction of phenolic compounds from wood, plants or wastewater effluents.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
60
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Extraction of Phenolic Compounds with Aqueous Two Phase Systems
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Co-author in
Tomé, L.I.N.; Dominguez-Perez, M; Cláudio , A.F.M; Freire, M.G.; Marrucho,
I.M.; Cabeza, O.,and Coutinho, J.A.P., On the Interactions between Amino Acids and Ionic
Liquids in Aqueous Media. Journal of Physical Chemistry B, 2009. 113(42): p. 13971-
13979.
Louros, C.L.S; Cláudio , A.F.M; Neves ,C.M.S.S.; Freire, M.G.; Marrucho, I.M.;
Pauly, J.; and Coutinho, J.A.P., Extraction of Biomolecules Using Phosphonium-Based
Ionic Liquids + K3PO4 Aqueous Biphasic Systems. International Journal of Molecular
Sciences, 2010. 11(4): p. 1777-1791
Cláudio, A.F.M.; Freire ,M.G.; Freire, C. S. R.; Silvestre, A. J. D.and Coutinho,
J.A.P. Extraction of Vanillin using Ionic-Liquid-Based Aqueous Two-Phase Systems,
Separation and Purification Technology, 2010, accepted for publication.
Neves, C. M. S. S.; Batista, M. L. S.; Cláudio, A. F. M.; Santos, L. M. N. B. F.;.
Marrucho, I. M; Freire, M G. and Coutinho, J A. P., Thermophysical Properties and Water
Saturation of [PF6]-based Ionic Liquids, Journal of Chemical & Engineering Data, 2010,
accepted for publication.
Cláudio, A. F. M.; Soto, A. M.; Freire, M.G. and Coutinho, J.A.P., Extraction of
gallic acid using Ionic-Liquid-Based Aqueous Two-Phase Systems, in preparation.
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
70
A.1. Calibration curve for vanillin
In order to know the amount of vanillin in each phase, it is necessary to draw a calibration curve.
For this, 7 standards of different known concentrations were prepared (Table A 1) from the initial
stock solution ([Van] = 0.513 g·dm-3
). Absorbance was measured at a wavelength of 280 nm.
Table A 1: Concentration of vanillin and respective absorbance at λ = 280 nm.
Standard [Van] / (g·dm-3
) Abs
1 2.57 × 10-2
1.694
2 2.05 × 10-2
1.377
3 1.03 × 10-2
0.707
4 5.13 × 10-3
0.372
5 4.10 × 10-3
0.281
6 6.16 × 10-3
0.432
7 8.21 × 10-3
0.570
The calibration curve obtained is displayed in Figure A1.
Figure A 1: Calibration curve for vanillin at λ = 280 nm.
Abs = 67.055 × [vanillin]R2 = 0.9986
0.0
0.5
1.0
1.5
2.0
0.00 0.01 0.02 0.03
Ab
s
[Vanillin] / (g.dm-3)
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
71
A.2. Calibration curve for gallic acid
To quantify gallic acid in each of the aqueous phases it was established the respective calibration
curve. As a result, 7 standards of different known concentrations were prepared (Table A 2) from
the initial stock solution ([GA] = 0.521 g·dm-3
). Absorbance was measured at a wavelength of 262
nm.
Table A 2: Concentration of gallic acid and respective absorbance at λ = 262 nm.
standard [GA] / (g·dm-3
) Abs
1 2.08×10-3
0.095
2 2.61×10-3
0.113
3 3.47×10-3
0.157
4 5.21×10-3
0.227
5 1.04×10-2
0.474
6 1.56×10-2
0.683
7 2.08×10-2
0.907
The calibration curve obtained is displayed in Figure A2.
Figure A 2: Calibration curve for gallic acid at λ = 262 nm.
Abs = 43.877 [gallic acid]
R² = 0.9994
0.0
0.5
1.0
0.00 0.01 0.02 0.03
Ab
s
[Gallic acid] / (g. dm-3)
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
73
Experimental data for the vanillin partition coefficients, density and
viscosity
Table B 1: Experimental weight fraction composition and partition coefficients of vanillin in ILs +
K3PO4 ATPS at 298.15 K.
IL + K3PO4 + water
system
Weight fraction composition / wt % KVan ± σa
IL K3PO4
[C2mim]Cl 24.83 15.28 36.49 ± 0.61
[C4mim]Cl 25.00 15.02 44.98 ± 0.10
[C6mim]Cl 25.66 14.62 49.59 ± 1.12
[C7mim]Cl 24.61 14.88 42.39 ± 1.35
[C10mim]Cl 24.80 15.33 2.72 ± 0.22
[amim]Cl 24.75 15.86 36.45 ± 1.60
[OHC2mim] 40.66 14.92 22.95 ± 0.12
[C7H7mim]Cl 24.54 14.78 44.18 ± 1.34
[C4mim][CH3CO2] 24.63 14.88 6.64 ± 0.54
[C4mim][N(CN)2] 24.63 14.77 31.87 ± 3.97
[C4mim]Br 24.74 15.55 25.66 ± 0.40
[C4mim][CH3SO3] 24.82 15.23 6.94 ± 0.05
[C4mim][CF3SO3] 24.67 15.16 9.75 ± 1.34
[C4mim][CH3SO4] 25.05 15.16 22.74 ± 0.55
astandard deviation
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
74
Table B 2: Experimental weight fraction composition and partition coefficients of vanillin in IL +
K3PO4 ATPS as a function of temperature.
T / K Weight fraction composition / wt % KVan± σa
IL K3PO4
[C4mim]Cl
288.15 24.64 15.06 21.46 ± 0.01
298.15 25.00 15.02 44.98 ± 0.10
308.15 24.68 14.94 45.97 ± 0.37
318.15 25.02 15.08 30.90 ± 0.47
328.15 24.98 14.99 19.26 ± 0.42
[C4mim][CH3SO4]
288.15 24.90 15.20 15.27 ± 1.12
298.15 25.05 15.16 22.74 ± 0.55
308.15 24.76 15.70 22.03 ± 0.49
318.15 24.71 15.06 17.35 ± 0.74
328.15 24.89 15.07 14.63 ± 1.07
[C7H7mim]Cl
288.15 24.90 15.28 29.25 ± 2.35
298.15 24.54 14.78 44.19 ± 1.34
308.15 24.59 14.76 30.52 ± 0.15
318.15 25.15 14.94 27.09 ± 0.20
328.15 24.55 14.96 26.99 ± 0.90
[amim]Cl
288.15 24.80 14.90 33.74 ± 0.53
298.15 24.76 15.86 36.45 ± 1.60
308.15 24.77 14.95 21.83 ± 0.64
318.15 24.78 15.42 22.03 ± 0.01
328.15 24.96 15.16 13.55 ± 0.13 astandard deviation
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
75
Table B 3: Experimental weight fraction composition and partition coefficients of vanillin in IL +
K3PO4 ATPS as a function of the initial vanillin concentration.
IL + K3PO4 + Water system with
different concentrations
Weight fraction composition / wt % KVan± σa
IL K3PO4
[C4mim]Cl
0.5 g·dm-3
= 3.3 × 10-3
mol·dm-3
25.05 15.14 33.09 ± 0.49
1.0 g·dm-3
= 6.6 × 10-3
mol·dm-3
25.00 15.20 44.98 ± 0.10
2.5 g·dm-3
= 1.6 × 10-2
mol·dm-2
24.73 15.48 57.02 ± 7.26
5.0 g·dm-3
= 3.3 × 10-2
mol · dm-2
25.05 15.00 88.30 ± 15.50
7.5 g·dm-3
= 4.9 × 10-2
mol · dm-2
24.96 14.99 98.08 ± 1.00
[C4mim][CH3SO4]
0.5 g·dm-3
= 3.3 × 10-3
mol·dm-3
25.02 15.19 17.97 ± 1.28
1.0 g·dm-3
= 6.6 × 10-3
mol·dm-3
25.05 15.16 22.74 ± 0.55
2.5 g·dm-3
= 1.6 × 10-2
mol·dm-2
24.86 15.30 35.04 ± 4.03
5.0 g·dm-3
= 3.3 × 10-2
mol · dm-2
24.14 15.75 36.19 ± 1.65
[C7H7mim]Cl
0.5 g·dm-3
= 3.3 × 10-3
mol·dm-3
24.95 14.97 21.31 ± 0.97
1.0 g·dm-3
= 6.6 × 10-3
mol·dm-3
24.54 14.78 44.19 ± 1.34
2.5 g·dm-3
= 1.6 × 10-2
mol·dm-2
24.94 15.07 60.00 ± 2.86
5.0 g·dm-3
= 3.3 × 10-2
mol · dm-2
24.61 14.71 79.58 ± 0.24
7.5 g·dm-3
= 4.9 × 10-2
mol · dm-2
24.86 14.90 98.69 ± 9.12
astandard deviation
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
76
Table B 4: Density and viscosity dependence on temperature of the IL-rich phase (Top phase, T)
and salt-rich phase (Bottom phase, B) for systems composed by chloride-based ILs + K3PO4 + H2O
equilibrated at 298.15 K.
T / K IL / wt % K3PO4 / wt % ηT / mPa.s ηB / mPa.s ρT / g.cm-3
ρB / g.cm-3
[C2mim]Cl + water + K3PO4
298.15
24. 58 15.92
2.9785 4.2829 1.0951 1.3857
308.15 2.3209 3.4014 1.0896 1.3796
318.15 1.8597 2.7726 1.0840 1.3734
328.15 1.5261 2.3107 1.0781 1.3671
[C4mim]Cl + water + K3PO4
298.15
24.80 15.46
3.6868 3.6160 1.0725 1.3609
308.15 2.7839 2.8889 1.0664 1.3549
318.15 2.1767 2.3672 1.0602 1.3488
328.15 1.7540 1.9775 1.0538 1.3427
[C6mim]Cl + water + K3PO4
298.15
25.11 14.99
5.3025 2.8001 1.0597 1.3138
308.15 3.9496 2.2715 1.0536 1.3078
318.15 3.0351 1.8827 1.0476 1.3019
328.15 2.3933 1.5901 1.0413 1.2959
[C7mim]Cl + water + K3PO4
298.15
24.84 15.59
10.208 2.6509 1.0515 1.3012
308.15 7.3087 2.1566 1.0455 1.2953
318.15 5.3741 1.7924 1.0393 1.2895
328.15 4.0472 1.5179 1.0329 1.2835
[C10mim]Cl + water + K3PO4
298.15
24.18 16.54
14.187 2.5565 1.0782 1.2565
308.15 10.7300 2.0785 1.0721 1.2507
318.15 8.3817 1.7303 1.0658 1.2449
328.15 6.6833 1.4676 1.0594 1.2389
[C7H7mim]Cl + water + K3PO4
298.15
25.09 14.97
4.1457 2.6911 1.1172 1.2939
308.15 3.1450 2.1835 1.1113 1.2880
318.15 2.4672 1.8106 1.1053 1.2821
328.15 1.9919 1.5302 1.0990 1.2761
[amim]Cl + water +K3PO4
298.15 2.9621 3.6759 1.1002 1.3596
308.15 24.98 15.13 2.2977 2.9377 1.0945 1.3535
318.15 1.8362 2.4095 1.0887 1.3475
328.15 1.5055 2.018 1.0827 1.3413
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
77
[OHC2mim]Cl + water + K3PO4
298.15
39.64 15.76
6.2735 9.2536 1.1832 1.5265
308.15 4.7395 6.9621 1.1779 1.5201
318.15 3.7035 5.4289 1.1727 1.5138
328.15 2.9719 4.359 1.1675 1.5075
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
78
Table B 5: Density and viscosity dependence on temperature of the IL-rich phase (Top phase, T)
and salt-rich phase (Bottom phase, B) for systems composed by [C4mim]-based ILs + K3PO4 + H2O
equilibrated at 298.15 K.
T / K IL / wt % K3PO4 / wt % ηT / mPa.s ηB / mPa.s ρT / g.cm-3
ρB / g.cm-3
[C4mim]Cl + water + K3PO4
298.15
24.80 15.46
3.6868 3.616 1.0725 1.3609
308.15 2.7839 2.8889 1.0664 1.3549
318.15 2.1767 2.3672 1.0602 1.3488
328.15 1.754 1.9775 1.0538 1.3427
[C4mim]Br + water + K3PO4
298.15
24.95 15.06
3.2282 2.7799 1.1496 1.3046
308.15 2.4675 2.2463 1.1432 1.2986
318.15 1.9501 1.8565 1.1368 1.2927
328.15 1.5839 1.5644 1.1299 1.2866
[C4mim][CH3SO4] + water + K3PO4
298.15
25.04 14.97
3.4649 2.8726 1.1254 1.2854
308.15 2.6485 2.3089 1.119 1.2796
318.15 2.0936 1.9023 1.1125 1.2737
328.15 1.7000 1.5985 1.1059 1.2677
[C4mim][CH3SO3] + water + K3PO4
298.15
24.11 16.49
3.6729 3.7196 1.1230 1.3459
308.15 2.7913 2.9666 1.1168 1.3397
318.15 2.1968 2.4258 1.1105 1.3335
328.15 1.7773 2.0240 1.1041 1.3273
[C4mim][CF3SO3] + water + K3PO4
298.15
24.98 15.02
8.0471 2.0015 1.2169 1.2291
308.15 5.9335 1.6372 1.2115 1.2208
318.15 4.5461 1.3656 1.2060 1.2125
328.15 3.5951 1.1594 1.1996 1.2041
[C4mim][CH3CO2] + water + K3PO4
298.15
24.98 14.93
3.7596 4.7622 1.1087 1.3830
308.15 2.8228 3.7208 1.1027 1.3774
318.15 2.2007 2.988 1.0966 1.3718
328.15 1.7677 2.4591 1.0903 1.3658
[C4mim][N(CN)2] + water + K3PO4
298.15
24.96 15.10
3.6187 2.3530 1.0366 1.2542
308.15 2.7869 1.9138 1.0297 1.2485
318.15 2.2100 1.5907 1.0229 1.2426
328.15 1.7969 1.3469 1.0159 1.2366
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
80
van’t Hoff plots
Figure C 1: van’t Hoff plot of ln(KVan) versus inverse absolute temperature for
[C4mim]Cl and [C4mim][CH3SO4] systems.
Figure C 2: van’t Hoff plot of ln(KVan) versus inverse absolute temperature for [C7H7mim]Cl and
[amim]Cl systems.
[C4mim]Cl [C4mim][CH3SO4]
y = 1.5181x - 1.9159
y = 2.8458x - 5.5936
0
1
2
3
4
5
3.0 3.1 3.2 3.3 3.4
ln(K
Va
n)
1 / T [103 K-1]
[C7H7mim]Cl [amim]Cl
y = 1.5859x - 1.6203
y = 2.8975x - 6.1702
0
1
2
3
4
5
3.0 3.1 3.2 3.3 3.4
ln(K
Van)
1 / T [103 K-1]
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
82
Experimental binodal curve mass fraction compositions
Table D 1: Experimental binodal curve and mass fraction compositions for the system IL +
Na2SO4 + H2O at 298 K.
[C4mim]Br Mr = 219.12 [C7mim]Cl Mr = 216.58 [C8mim]Cl Mr = 230.78
100 w1 100 w2 100 w1 100 w2 100 w1 100 w2
54.039 2.245 49.164 4.570 48.964 4.640
50.037 2.943 46.036 5.467 46.616 5.599
46.376 3.518 42.213 6.747 42.090 7.098
42.669 4.363 36.094 8.979 38.460 8.185
40.734 4.920 32.220 10.669
39.139 5.343 30.624 11.230
34.186 7.139
Table D 2: Experimental binodal curve and mass fraction compositions for the system IL +
Na2SO4 + H2O at 298 K.
[C4mim][CF3SO3] Mr = 288.29 [C7H7mim]Cl Mr = 208.69
100 w1 100 w2 100 w1 100 w2
55.957 1.742 15.200 6.486 52.119 3.459
49.416 2.184 14.489 6.971 46.554 4.804
42.662 2.545 12.249 7.804 40.895 6.141
34.874 3.111 11.548 8.233 37.642 7.216
32.697 3.374 11.010 8.508 33.469 8.885
30.563 3.644 10.031 9.111
29.011 3.720 9.383 9.590
27.770 3.993 8.829 10.178
26.080 4.116 7.781 11.017
24.786 4.326 7.124 11.627
23.475 4.361 6.758 12.100
22.857 4.509 5.826 13.242
21.694 4.761 5.235 13.992
20.616 5.001 4.646 14.956
19.258 5.361 4.297 15.492
17.943 5.642 3.879 16.222
16.705 5.914 3.503 16.992
16.043 6.150
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
83
Table D 3: Experimental binodal curve and mass fraction compositions for the system IL +
Na2SO4 + H2O at 298 K.
[C4mim][TOS] Mr = 310.42
100 w1 100 w2 100 w1 100 w2 100 w1 100 w2
55.113 2.197 19.397 11.255 11.239 14.492
52.339 2.637 19.050 11.426 10.871 14.705
49.096 2.892 18.596 11.531 10.518 14.874
47.612 3.188 18.292 11.668 10.371 14.978
45.586 3.412 17.971 11.814 10.175 15.076
44.347 3.743 17.611 12.010 10.010 15.244
43.047 4.083 17.409 12.038 9.880 15.272
40.476 4.722 17.182 12.123 9.662 15.403
38.394 5.230 16.891 12.282 9.453 15.540
36.588 5.600 16.415 12.469 9.254 15.664
35.800 5.793 16.224 12.492 9.037 15.805
35.029 6.008 15.925 12.663 8.619 16.041
34.144 6.317 15.735 12.708 8.326 16.232
33.392 6.527 15.576 12.744 8.095 16.367
32.564 6.722 15.416 12.805 7.905 16.481
31.945 6.934 15.174 12.936 7.528 16.753
31.286 7.103 15.006 12.972 7.282 16.904
30.643 7.271 14.792 13.091 6.957 17.135
30.008 7.396 14.579 13.207 6.621 17.415
29.402 7.572 14.360 13.312 6.282 17.651
28.845 7.747 14.230 13.329 6.077 17.823
28.312 7.915 13.999 13.472 5.963 17.927
26.539 8.557 13.879 13.496 5.751 18.148
25.875 8.877 13.686 13.595 5.454 18.389
25.520 8.940 13.382 13.720 5.253 18.597
24.958 9.155 13.258 13.751 5.101 18.727
24.438 9.372 13.157 13.797 4.919 18.887
24.047 9.498 12.971 13.907 4.830 18.997
23.591 9.644 12.854 13.933 4.644 19.210
22.808 9.888 12.692 14.030 4.339 19.514
22.245 10.169 12.526 14.122 4.087 19.777
21.207 10.492 12.247 14.097 3.834 20.156
20.842 10.630 12.133 14.133 3.621 20.379
20.465 10.777 11.768 14.237 3.433 20.652
20.058 10.996 11.466 14.366 3.283 20.802
19.751 11.086
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
84
Table D 4: Experimental binodal curve and mass fraction compositions for the system IL +
Na2SO4 + H2O at 298 K.
[C4mim][N(CN)2] Mr = 205.26
100 w1 100 w2 100 w1 100 w2 100 w1 100 w2
59.251 0.599 18.934 9.451 6.790 16.014
54.869 1.101 18.450 9.610 6.549 16.284
51.855 1.614 17.981 9.775 6.431 16.386
47.900 1.966 17.765 9.848 6.320 16.525
45.730 2.355 14.851 11.048 6.138 16.701
43.248 2.673 14.693 11.088 5.950 16.907
41.487 3.022 14.471 11.236 5.749 17.110
39.764 3.322 14.196 11.335 5.319 17.916
38.715 3.649 14.063 11.367
37.412 3.904 13.923 11.410
35.952 4.171 13.717 11.571
34.708 4.418 13.462 11.652
33.951 4.670 13.165 11.809
32.711 4.942 12.856 11.949
31.958 5.187 12.657 12.036
31.263 5.369 12.421 12.151
30.331 5.489 12.274 12.262
29.697 5.708 11.913 12.463
29.112 5.874 11.740 12.511
28.518 6.109 11.604 12.599
27.970 6.317 11.387 12.720
27.152 6.463 11.165 12.837
26.597 6.661 10.965 12.953
25.517 7.044 10.554 13.290
25.039 7.217 10.260 13.381
24.580 7.433 10.117 13.426
24.157 7.579 10.015 13.506
23.747 7.710 9.819 13.641
23.333 7.849 9.624 13.790
22.959 8.034 9.370 13.967
22.275 8.267 9.047 14.168
21.612 8.501 8.893 14.261
20.968 8.706 8.710 14.426
20.680 8.790 8.321 14.722
20.373 8.873 8.082 14.868
19.829 9.060 7.843 15.085
19.565 9.148 7.451 15.400
19.312 9.240 7.125 15.694
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
85
Table D 5: Experimental binodal curve and mass fraction compositions for the system IL +
Na2SO4 + H2O at 298 K.
[C8py][N(CN)2] Mr = 258.36 [C2mim][CF3SO3]
Mr = 260.24
100 w1 100 w2 100 w1 100 w2 100 w1 100 w2
57.558 1.239 15.158 5.532 53.316 2.464
43.629 2.068 15.007 5.564 49.414 3.075
39.144 2.265 14.894 5.646 44.443 3.783
33.813 2.626 14.708 5.713 43.102 3.971
31.218 2.919 14.503 5.761 41.236 4.388
28.714 3.126 14.366 5.775 39.598 4.803
27.437 3.349 14.246 5.815 38.152 5.206
26.157 3.450 14.119 5.851 36.404 5.693
25.445 3.479 13.914 5.865 34.797 6.112
25.118 3.579 13.850 5.920 33.440 6.455
24.499 3.619 13.677 5.986 32.291 6.768
24.054 3.692 13.358 6.037 30.753 7.254
23.770 3.739 13.244 6.083 29.663 7.553
23.281 3.821 13.109 6.125 28.334 8.005
22.810 3.883 12.893 6.229 27.493 8.240
22.215 3.971 12.617 6.301 26.666 8.506
21.786 4.041 12.408 6.423 25.586 8.888
21.447 4.109 11.815 6.574 24.893 9.102
20.057 4.378 11.590 6.749 23.973 9.430
19.746 4.518 11.010 6.896 23.318 9.598
19.335 4.530 10.783 6.976 22.517 9.907
19.043 4.586 10.488 7.154 21.925 10.074
18.807 4.702 9.947 7.513 21.142 10.351
18.363 4.719 9.491 7.571 20.381 10.643
18.054 4.862 9.273 7.718 19.550 10.997
17.623 4.891 8.923 7.888 18.920 11.214
17.432 4.987 8.521 8.084 18.138 11.560
17.171 5.013 8.225 8.280 17.601 11.756
17.042 5.079 7.965 8.441 17.010 12.015
16.860 5.118 7.761 8.494 16.426 12.279
16.703 5.133 7.288 8.787 15.778 12.605
16.548 5.193 6.801 9.184 15.186 12.893
16.435 5.218 6.045 9.881 14.698 13.116
16.328 5.278 5.523 10.302 14.093 13.452
16.171 5.292 5.229 10.548 12.572 14.329
16.038 5.346 4.734 11.083 11.590 14.898
15.836 5.343 4.281 11.658 10.825 15.358
15.719 5.370 3.829 12.274 9.410 16.401
15.471 5.586 3.403 12.947 8.169 17.421
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
86
Table D 6: Experimental binodal curve and mass fraction compositions for the system IL +
Na2SO4 + H2O at 298 K.
[C7H7mim][C2H5SO4] [C4mim][CH3SO4] [C4mim][C2H5SO4]
Mr = 298.36 Mr = 250.32 Mr = 265.35
100 w1 100 w2 100 w1 100 w2 100 w1 100 w2
53.325 2.856 53.318 3.127 56.885 1.750
50.037 3.461 46.121 4.436 41.987 4.250
46.107 4.032 40.626 5.431 39.097 5.030
44.440 4.251 38.709 5.907 36.809 5.847
43.172 4.520 37.260 6.521 34.510 6.438
41.480 5.053 34.647 7.461 33.315 6.880
40.560 5.306 33.481 7.965 30.874 7.808
39.592 5.515 32.400 8.401 29.510 8.403
38.323 5.862 31.375 8.818 27.694 9.197
37.513 6.048 30.064 9.483 26.378 9.856
36.273 6.435 28.620 10.203 25.056 10.566
33.795 7.444 27.569 10.706 22.221 12.114
32.349 7.958 26.467 11.281 18.628 14.283
30.732 8.520 24.877 12.146 17.313 15.024
29.664 8.897 23.222 13.149 15.724 16.019
28.767 9.163 22.076 13.776 14.248 16.951
27.353 9.743 20.872 14.489 12.380 18.173
26.479 10.055 19.588 15.258
25.609 10.373 18.421 15.985
24.307 10.924 17.405 16.602
23.738 11.076 16.376 17.251
22.720 11.495 15.476 17.820
21.949 11.805 14.471 18.498
20.877 12.255 13.697 19.017
20.147 12.574 12.642 19.770
19.486 12.846 11.653 20.482
18.838 13.117 9.759 21.980
18.180 13.320
17.289 13.752
16.819 13.952
16.220 14.228
15.662 14.468
15.092 14.739
14.573 14.996
13.458 15.634
12.753 15.986
12.249 16.246
10.512 17.270
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
88
Experimental data of TL
Table E1: Weight fraction compositions for the coexisting phases at the TLs, and respective values
of α and TLL.
IL + Na2SO4 + Water
system
Weight fraction composition / wt% α TLL
YT XT YM XM YB XB
[C2mim][CF3SO3] 71.67 0.80 24.88 15.28 2.22 22.30 0.33 72.70
[C4mim]Br 37.42 5.91 25.66 15.03 10.57 26.73 0.56 33.98
[C4mim][TOS] 55.58 2.07 20.24 15.59 1.54 22.74 0.35 57.86
74.68 0.54 33.65 14.24 0.61 25.26 0.45 78.09
[C4mim][CH3SO4] 40.10 5.85 24.99 14.99 6.20 26.37 0.55 39.62
[C4mim][C2H5SO4] 36.82 5.74 25.71 15.36 0.47 37.19 0.69 48.07
[C4mim][N(CN)2] 59.77 0.79 24.94 15.14 0.37 25.26 0.41 64.25
[C4mim][CF3SO3] 33.31 3.16 24.46 14.77 2.91×10-14
46.84 0.73 54.93
[C7mim]Cl 31.29 10.89 25.03 14.85 1.43 29.75 0.79 35.32
[C8mim]Cl 36.91 8.91 27.24 14.89 0.28 31.58 0.74 43.08
[C7H7mim]Cl 33.63 8.77 27.90 12.13 10.22 22.49 0.51 27.13
36.06 7.84 24.62 15.05 5.82 26.90 0.62 35.74
[C7H7mim][C2H5SO4] 55.86 2.34 24.92 15.18 2.22 24.60 0.42 58.08
60.95 1.70 31.82 14.26 0.91 27.58 0.51 65.39
[C8py][N(CN)2] 69.11 0.93 24.57 15.36 0.31 23.23 0.35 72.32
73.41 0.82 31.46 14.07 0.26 23.92 0.51 76.71 astandard deviation
Extraction of Phenolic Compounds with Aqueous Two Phase Systems
90
Experimental data for the gallic acid partition coefficients
Table F 1: Experimental weight fraction composition and partition coefficients of gallic acid in ILs
+ Na2SO4 ATPS at 298.15 K.
IL + Na2SO4 +
water system
Weight fraction composition / wt % KGA ± σ
a
IL Na2SO4
[C2mim][CF3SO3] 24.88 15.28 10.25 ± 0.01
[C4mim][CF3SO3] 24.46 15.37 20.73 ± 1.33
[C4mim]Br 24.99 15.04 18.12 ± 0.10
[C4mim][N(CN)2] 24.94 15.14 21.23 ± 0.14
[C4mim][CH3SO4] 24.99 14.99 20.84 ± 0.95
[C4mim][C2H5SO4] 25.71 15.36 29.58 ± 1.19
[C7mim]Cl 25.03 14.85 21.94 ± 1.57
[C8mim]Cl 26.50 15.06 19.39 ± 0.19 astandard deviation
Table F 2: Experimental weight fraction composition and partition coefficients of gallic acid in ILs
+ K3PO4 ATPS at 298.15 K.
IL + K3PO4 +
water system
Weight fraction composition / wt % KGA ± σ
a
IL K3PO4
[C2mim][CF3SO3] 24.92 15.03 0.576 ± 0.191
[C4mim][CF3SO3] 25.09 15.00 0.175 ±0.007
[C7mim]Cl 24.74 14.96 8.162 ±0.362 astandard deviation
Table F 3: Experimental weight fraction composition and partition coefficients of gallic acid in ILs
+ K2HPO4/ KH2PO4 ATPS at 298.15 K.
IL +
K2HPO4/KH2PO4
+ water system
Weight fraction composition / wt % KGA ± σ
a
IL K2HPO4/KH2PO4
[C2mim][CF3SO3] 25.01 15.00 0.909 ± 0.052
[C4mim][CF3SO3] 24.99 15.13 0.763± 0.003
30.01 15.05 4.253 ± 0.172
[C7mim]Cl 29.97 15.00 11.586 ± 1.000 astandard deviation